Nanofibrous materials as drug, protein, or genetic release vehicles

ABSTRACT

The present invention is a bioactive, nanofibrous material construct which is manufactured using a unique electrospinning perfusion methodology. One embodiment provides a nanofibrous biocomposite material formed as a discrete textile fabric from a prepared liquid admixture of (i) a non-biodegradable durable synthetic polymer; (ii) a biologically active agent; and (iii) a liquid organic carrier. These biologically-active agents are chemical compounds which retain their recognized biological activity both before and after becoming non-permanently bound to the formed textile material; and will become subsequently released in-situ as discrete freely mobile agents front the fabric upon uptake of water from the ambient environment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/293,481 (filedJun. 2, 2014), which is; a continuation-in-part of U.S. Ser. No.13/303,319, filed Nov. 23, 2011 (now issued U.S. Pat. No. 8,771,582),which is a continuation-in-part of U.S. Ser. No. 12/954,829, filed Nov.26, 2010 (now issued U.S. Pat. No. 8,691,543), which claims priority toU.S. Provisional Application 61/264,440, filed Nov. 25, 2009. Theaforementioned U.S. Ser. No. 13/303,319 is also a continuation-in-partof U.S. Ser. No. 11/366,165, filed Mar. 2, 2006 (abandoned) which is acontinuation-in-part of U.S. Ser. No. 11/211,935, filed Aug. 25, 2005(now issued U.S. Pat. No. 7,413,575), which claims priority to U.S.Provisional Application 60/658,438 (filed Mar. 4, 2005). Theabove-identified patent applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The instant invention provides a variety of non-biodegradable, formedfabric materials, articles, and devices suitable for the in-situdelivery of many different biologically-active agents. The disclosurealso offers a wide range of fabricated nanofibrous textiles havingvarying and diverse individual biologic properties, or combinationsthereof; and provides medical products which are resistant to breakageand tearing as well as demonstrate a specifically desired localizedeffect such as resistance to infection-properties which will aid inreducing both the morbidity and mortality of a person afflicted with aninjury or ailment.

BACKGROUND

There are over 13 million medical articles and devices utilized annuallyin the United States for prophylactic and/or therapeutic treatment.These items range in sophistication from simple devices such as herniarepair mesh, wound dressings and catheter cuffs—to more compleximplantable devices such as the total implantable heart, leftventricular assist devices and prosthetic arterial grafts. Althoughutilization of these medical articles and devices has improved thehealth and quality of life for the patient population as a whole, thein-vivo application of all such medical implements are prone to twomajor kinds of complications: injection and incomplete/non-specificcellular healing.

In general, regardless of the particular causative agent, infectionremains one of the major complications associated with utilizingbiomaterials, with the clinical infection occurring at either acute ordelayed time periods after in-vivo use or implantation of the medicalarticle or device. Today, surgical site infections account forapproximately 14-16% of the 2.4-million nosocomial infections in theUnited States, and result in an increased patient morbidity andmortality. The inherent bulk properties of various biomaterials thatcomprise these articles and devices typically provide a milieu forinitial bacterial/fungus adhesion with subsequent biofilm production andgrowth.

Similarly, unregulated cellular growth affects various medical devicessuch as stents and vascular grafts. Occlusion rates for diseased bloodvessels after placement of a bare metallic stent (restenosis) have beenreported as high as 27%, a significant problem based on the 1.1 millionstents annually implanted. Moreover, since the currently availablebiomaterials in these medical articles and devices are typicallycomprised of foreign polymeric compounds, these biomaterials do notemulate the multitude of dynamic biologic and healing processes thatoccur in normal tissue; and consequently, the cellular componentsnormally present within native living tissue are not available forcontrolling and/or regulating the reparative process. Thus, the searchcontinues today for novel biomaterials (such as drug releasingbiomaterials) that would direct or enhance some of the normal healingprocesses of native tissue, and would decrease patient morbidity andmortality rates.

Currently, drug delivery from a majority of implantable medical devicessuch as stents is achieved via the coating/scaling of a device orscaffold with a biodegradable polymer composition which serves as a drugreservoir. There are several potential problems with utilizing thissystem in that: (1) polymer coating onto the device can be inconsistent,resulting in areas with minimum/no localized drug release; (2) polymercoating efficiency can be limited based on the device design orcomposition of the base material; (3) drug release is dependent onbiodegradation of the polymer reservoir, resulting in inconsistent drugrelease; and (4) application of the exogenous polymer can have adverseeffects on tissue/organ healing or upon the biocompatibility (i.e.increasing thrombogenecity) of the original implant.

Electrospinning provides a technique for making nanofibrous materialsubstrates. Electrospinning to produce nanoscale fibers, fabricationsand textiles, however, is still a manufacturing technique in need offurther development and refinement. Utilization of electrospinning as atechnique to synthesize various nanofibrous materials from polymers suchas polyurethane, polyvinyl alcohol (or “PVA”), poly(lactic glycolic)acid (or “PLGA”), nylon, and polyethylene oxide has been investigatedfor several decades (see for example Subbiah et al., “Electrospinning OfNanofibers”, J. Applied Polymer Sci. 96:557-569 (2005).

While inclusion of bioactive agents has been accomplished for severalother polymers (such as polyurethane, PLGA, alginate and collagen), theelectrospinning technique has not been realized for polyethyleneterephthalate (“PET”), or “polyester” as understood generally in textilecircles, until recently. Since then. Ma et al. was able to electrospinpolyethylene terephthalate using a melt-spinning technology (see Ma Z,Kotaki M, Yong T, He W, Ramakrishna S., “Surface engineering ofelectrospun polyethylene terephthalate (PET) nanofibers towardsdevelopment of a new material for blood vessel engineering”,Biomaterials 26:2527 (2005)). However, the Ma et al. reported techniquerequires a surface modification in which formaldehyde and severalcross-linkers were utilized post-spinning subsequently to incorporategelatin, owing to the high temperatures employed in their manufacturingprocess. These modification procedures are and remain a major issuebecause of their high temperature requirements and the consequentialfailure of the protein (or other temperature sensitive agent) tomaintain its characteristic biological activity throughout the materialfabrication process.

Accordingly, despite all these developments to date, there remains arecognized and continuing need for further improvements in the making ofmedical devices and articles comprised of nanofibrous materials whichwould demonstrate adequate physical strength characteristics anddurability as fabricated items, and which would serve as biomedicalconstructs formed of fibrous materials having demonstrable biologicallyactive properties. All such improvements in the making and/orpreparation of such nanofibrous materials and articles would be readilyseen as a major advantage and outstanding benefit in the medical field.

SUMMARY OF THE INVENTION

The present invention is a major advance in the development ofbiomedical materials, devices and constructs. Accordingly, the inventionhas multiple aspects, some of which may be defined as follows.

A first aspect provides a method for forming a fabricated textilesuitable for use as a medical article. The method includes the steps ofdissolving a non-biodegradable polymer and a pre-chosenbiologically-active agent in an organic solvent at an ice-coldtemperature. Once dissolved, the admixture is permitted to warm beforeelectrospinning at room temperature to form the fabricated textile.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is disclosed with reference to the accompanyingdrawings, wherein:

The present invention may be more easily understood and more readilyappreciated when taken into conjunction with the accompanying drawing,in which:

FIG. 1 is an illustration of the chemical structure of Ciprofloxacin;

FIG. 2 is an illustration of the chemical structure of Diflucan;

FIG. 3 is an illustration of the chemical structure of Paclitaxel;

FIG. 4 is a an illustration of the apparatus for performing theelectrospinning methodology;

FIG. 5A and FIG. 5B are scanning electron microphotographs of a nPET(electrospun polyethylene terephthalate) textile segment showing thediameter size of the fibers within the nanofibrous material;

FIG. 6 is an overhead view of the UV illumination differences betweennPET segments, nPET-Cipro segments, and nPET-Diflucan segments;

FIG. 7 is a graph showing the release profile of Cipro from nPET-Ciprosegments over time;

FIG. 8 is a graph showing the release profile of Diflucan fromnPET-Diflucan segments over time;

FIG. 9 is a an overhead view of the inhibitions zone againstStaphylococcus aureus streaked onto agar plates;

FIG. 10 is a graph showing the antimicrobial activity of nPET-Ciprosegments over time;

FIG. 11 is a graph showing the anti-fungal activity of nPET-Diflucansegments against varying concentrations of Candida albicans; and

FIG. 12 illustrates an overhead view of a flat sheet of electrospuntextile fabric.

Corresponding reference characters indicate corresponding partsthroughout the several views. The examples set out herein illustrateseveral embodiments of the invention but should not be construed aslimiting the scope of the invention in any manner.

DETAILED DESCRIPTION

Disclosed in this specification is a bioactive, nanofibrous materialconstruct which is manufactured either in tubular or flat sheet formusing an unique electrospinning perfusion methodology. One particularembodiment provides a nanofibrous biocomposite material formed as adiscrete textile fabric from a prepared liquid admixture of (i) abiodurable synthetic polymer; (ii) a biologically active agent; and(iii) a liquid organic carrier. The prepared liquid admixture of diversecompositions is employed in a novel electrospinning perfusion process toform an agent-releasing textile comprised of nanofibrous material, whichin turn, can serve as the antecedent precursor and tangible workpiecefor subsequently making the desired medical article or device suitablefor use in-vivo. Prior art medical devices generally includes anunderlying non-polymeric support (e.g. scaffold, stent, etc) and coatthe support with a biodegradable polymer and then soaks the resultingcoated support in a biologically-active agent to embed the agent in thepolymer. In contrast, the medical devices of the present invention arediscrete articles that omit the underlying scaffold and the medicaldevices consist essentially of a non-biodegradable polymer that has thebiologically-active agent embedded therein. The materials of the presentinvention have mechanical properties which are sufficient to permit themanufacturer to omit the scaffolds that were previously required by theprior art.

After the agent-releasing textile has been fabricated as a discretearticle, one or more pre-chosen biologically-active agents will havebecome non-permanently immobilized and releasably bound to the tangiblenanofibrous material of the fabricated textile. These non-permanentlyimmobilized biologically-active agents are well established chemicalcompounds which retain their recognized biological activity both beforeand after becoming impermanently (i.e., temporarily or reversibly) boundto the textile fabric; and will become subsequently released in-situ anddirectly delivered into the ambient environment as discrete mobileentities when the textile fabric lakes up any fluid—i.e., any aqueous ororganic based liquid. Accordingly, via the transitory immobilization ofone or more biologically active molecules to the nanofibrousbiocomposite material, the agent-releasing textile is very suitable forinclusion and use in-vivo as a clinical/therapeutic construct.

The present electrospinning perfusion method of making agent-releasingnanofibrous textiles provides several major advantages and desirablebenefits to the commercial manufacturer as well as to the physician andsurgeon. Among these are the following:

First, the manufacturing methodology comprising the present inventiondocs not utilize any immersion techniques and does not requiresubmerging the fabricated textile in any immersion baths, soaking tanks,or dipping pools for any purpose. Rather, the methodology preferablyutilizes the unique technique of electrospinning perfusion as amanufacturing method in order to blend a synthetic substance and abiologically active agent of choice together as a fabricated textile.

Second, the electrospinning perfusion method of manufacture yields afabricated textile having particular characteristics. The fabricatedtextile is initially fashioned either as an elongated hollow tube havingtwo discrete open tubular ends and fixed inner and outer wall diameters;or as a flat or planar sheet of nanofibrous fabric. In either format,the fabricated textile can be folded, or twisted, and otherwisemanipulated to meet specific requirements of thickness, gauge, ordeniers; and can also be cut, split, tailored, and conformed to meetparticular shapes, configurations and patterns.

Third, the fabricated textile is a nanofibrous material compositecomprised of multiple fibers, has a determinable individual fiberthickness in or near the nanometer size range (typically less than 2microns), and presents a discernible fiber organization and distributionpattern. These fabricated textiles provide and demonstrate excellentsuture retention, burst strength, break strength, tear strength and/orbiodurability.

Fourth, the manufacturing method comprising the present inventionemploys limited heat and compression force to alter the exterior surfaceof the fabricated textile originally formed via the electrospinningperfusion technique. This exterior surface treatment portion of themanufacturing process is optional, but when employed, will produce ahighly desirable crimped exterior surface over the entire linear lengthof the fabricated textile article. A notable feature of this exteriorsurface treatment procedure is that the inner diameter size (typicallyless than 1 mm to not greater than about 30 mm, but can vary from theseparticular parameters) of the fabricated textile remains constant anduniform, despite the effects of the limited heating and compressiontreatment of the textile exterior surface.

Fifth, the biologically active agent will retain its characteristicbiological activity both before and after being temporarily bound to thenanofibrous material. The attributes and properties associated with thebiologically active agent of choice will co-exist with and be anintegrated feature of the resulting textile article at the time it isutilized.

The Agent-Releasing Nanofibrous Textile and its Role as an Antecedent inthe Making of a Prepared Medical Article or Device

The method of the present invention is directed in part to the making ofan agent-releasing textile, an antecedent article of manufacture, whichis then employed as a tangible workpiece to generate a subsequentlyprepared medical article or device suitable for use in-vivo. Anagent-releasing textile is a fabricated textile comprising nanofibrousmatter which has at least one biologically active agent immobilized ontoand/or within the material substance of the textile; and which, uponwetting, is then able to release the biologically active agent in-situand deliver it in a functionally operative form into the adjacent localarea or immediately surrounding environment. Such a prepared nanofibroustextile must provide and release at least one active chemicalcomposition, compound, or molecule which is active, functional andoperative either to influence and/or to initiate or cause a recognizablepharmacological effect or determinable physiological change in theliving cells, tissues and organs of the host patient. A fabricatedtextile is an article of manufacture which is comprised, in whole or inpart, of fibers arranged as a fabric. The fibers comprising thefabricated textile may be chosen from a diverse range of organicsynthetics, prepared polymer compounds, or naturally-occurring matter.In general, the fabricated textile is often prepared as a cloth orfabric; and may comprise a single fiber film, or a single layer offibrous matter; or exist as multiple and different deniers of fiberswhich are present in a range of varying thickness, dimensions, andconfigurations.

It will be appreciated that, after the agent-releasing nanofibroustextile has been manufactured and is present as a discrete entity, itcan optionally serve as a tangible workpiece in combination with otheritems and additional components and hardware to yield the desired endproduct, a clinically or therapeutically useful “medical article ordevice”. Thus, regardless of its true chemical composition/formulationor the particular mode of construction, the initially formed“agent-releasing textile” and the subsequently generated “medicalarticle or device” are directly and intimately related; and thus share anumber of specific qualities and characteristics in common. Thesemutually shared attributes include:

-   -   (i) Each agent-releasing textile is formed as an elongated        hollow tube having a determinable overall tubular length and two        open ends; has at least one internal lumen of determinable        volume which is co-incidental and coextensive with the internal        wall surface; and has at least one exterior wall surface which        is co-incidental and co-extensive with the outer wall        topography.    -   (ii) Each agent-releasing textile has a determinable length,        girth and depth of non-perforated fibrous material which can be        prepared to meet specific shapes, sizes and thicknesses of solid        matter;    -   (iii) Each agent-releasing textile can be employed either as a        configured tubular conduit whose internal lumen is usefully        employed for the conveyance of fluids in-situ, or,        alternatively, as a solid mass of nanofibrous material which        achieves its intended purpose without regard to or actual use of        the internal lumen then existing within the textile fabric.

By definitional requirement, the agent-releasing nanofibrous textile(optionally also the antecedent forerunner of each subsequentlygenerated medical article or device) is a non-woven material comprisedof discrete fibers. The nanofibrous composite material forming thetextile fabric has been electrospun from a liquid admixture and blendingin a liquid organic carrier of at least two different materials: asynthetic substance and a biologically active agent. This admixture oftwo diverse chemical compositions can be prepared in a wide range ofvarying ratios using a liquid organic carrier, followed by applicationof an electric current to create the biocomposite material

To illustrate the range and variety of compositions deemed suitable foruse as a blended mixture, a listing of suitable synthetic substances ispresented by Table 1 below. It will be noted that the listing of Table 1presents some exemplary synthetic substances long deemed suitable foruse as synthetic fibers. To complete the description. Table 2 lists someof the typical and more commonly available organic liquids which can beusefully employed alone and/or in blends as the liquid carriers.

TABLE 1 Illustrative Synthetic Substance Polymeric Fibers polyethyleneterephthalate; polybutylene terephthalate; polytrimethylene terephthlatePolyurethane; polyglycolic acid; polyamides, including nylons andaramids; Polytetrafluoroethylene; and mixtures of these substances Othersynthetic fiber compositions (using TFPIA generic fiber names) Acetate;Triacetate; Acrylic; Modacrylic; Olefin (Polypropylene, polyethylene,and other polyolefins); saran

TABLE 2 Representative Organic Liquid Carriers Hexafluoroisopropanol;Dimethylformamide; Dimethylsulfoxide; Acetonitrile; Acetone;Hexamethylphosphoric triamide; N,N-diethylacetamine;N-methylpyrrolidinone; Ethanol; 4-methylmorpholine-N-oxide monohydrate

At least some of the fibers comprising the textile fabric willdemonstrate a range of properties and characteristics, as follows.

1. The fibers constituting the agent-releasing textile (and thesubsequently generated medical article or device) will have ademonstrable capacity to take up water and/or aqueous liquids and/ororganic liquids and/or organic based liquids (with or without directwetting of the fibrous material). The mode or mechanism of action bywhich organic and aqueous fluids are taken up by the fibers of thetextile (and/or become wetted by the fluid) is technically insignificantand functionally meaningless.

Thus, among the different possibilities of fluid (aqueous and/ororganic) uptake are the individual alternatives of: absorption;adsorption; cohesion; adhesion; covalent bonding; non-covalent bonding;hydrogen bonding; miscible envelopment; molecule entrapment;solution-uptake between fibers; fiber wetting; as well as others welldocumented in the scientific literature. Any and/or all of these maycontribute to organic and/or aqueous fluid uptake in whole or in part.Which mechanism of action among these is actively in effect in anyinstance or embodiment is irrelevant.

2. By choosing a particular chemical formulation and/or desiredstereoscopic (or three-dimensional) structure for the syntheticsubstance of the fabrication, the resulting biologically active textilecan be prepared as a fabric having a markedly long functional durationand lifespan for in-vivo use. Accordingly, by choosing one or moredurable and highly resilient chemical compositions as the fibers ofchoice, textiles effective for many years' duration and utility may beroutinely made. All of these choices and alternatives are conventionallyknown and commonly used today by practitioners in this field.

It is also well recognized that some synthetic chemical compositions areavailable in a range of diverse formulations. As one example of a highlyresistant chemical composition having many alternative formulations arethe polyethylene terephthalates, of which one particular formulation issold under the trademark DACRON.

As is commonly known in this field, a range of differently formulatedpolyethylene terephthalates (or “PETs”) are known to exist and arecommercially available, each of these alternatives having a differentintrinsic viscosity [or “IV”, as measured in o-chlorophenol or “OCP”, at25° C.]. Typically, these differently formulated polyethyleneterephthalate compounds can vary from less than 0.6 dl/g [IV] to greaterthan 1 dl/g [IV]; yet each of these alternative polyethyleneterephthalate formulations can be dissolved in ice-cold 100%hexafluoroisopropanol. Thus, the electrospinning of appropriatelyprepared HFIP solutions containing any of such alternatively formulatedpolyethylene terephthalates will result in the fabrication ofnanofibrous textile fabrics which are capable of independent or combinedrelease of many diverse drugs, proteins and genetic materials.

3. The fibers comprising the agent-releasing textile (and thesubsequently generated medical article or device) can be prepared in avariety of organizations as a tangible structure. Thus, asconventionally recognized within the textile industry, the textilefabric may vary in size or thickness; and may optionally receive one ormore interior and/or exterior surface treatments to enhance particularattributes such as increased in-vivo biocompatibility or a greaterexpected time for functional operation and use in-vivo. All of theseorganizational variances are deemed to be routine matters which will beoptionally chosen and desirably used to meet particular medical needs orindividual patient requirements.

4. The fibers comprising the agent-releasing textile (and thesubsequently generated medical articles or devices) can be prepared tomeet the particulars of the intended in-vivo medical use circumstancesor the contingencies of the envisioned clinical/therapeutic application.Thus, the textile fabric can alternatively be prepared either as arelatively thin-walled biocomposite, or alternatively as a thick-walledmaterial; be produced as an elongated object having a diverse range ofdifferent outer diameter and inner diameter sizes; and be fashioned as arelatively inflexible or unyielding item or as a very flexible andeasily contorted length of matter.

B. The Choosing of an Appropriate Biologically Active Agent

A number of different biologically active agents can be beneficially andadvantageously utilized in tandem with the nanofibrous textile fabric.However, there are several minimal requirements and qualifications whichthe biologically active molecule-whatever its particular composition andformulation as a chemical compound, composition or molecule-mustdemonstrably provide in order to be suitable for use in the presentinvention. These are:

-   -   (i) The chosen agent must be capable of demonstrating its        characteristic biological activity before becoming temporarily        bound to and immobilized by the material substance of the        fabricated textile. This characteristic biological activity must        be well recognized and will constitute its ability/capacity to        function as an active mediator in-situ.    -   (ii) The particular agent immobilized upon or within the        material substance of the textile fabric must be capable of        demonstrating its characteristic biological activity (its        mediating capacity) after becoming immobilized and bound; and    -   (iii) The immobilized agent bound into the material substance of        the textile fabric will be released in-situ from the        non-biodegradable polymer and be delivered into the surrounding        local environment as a freely mobile molecule which retains its        characteristic biological activity (its mediating capacity) over        an extended period of time after the agent-releasing textile has        been utilized in-vivo and allowed to take up water.

In addition, since the primary medical application for the fabricatedtextile is expected to differ and vary extensively from one embodimentto another, it is intended that the characteristic biological propertiesof the chosen agent serve to aid, promote, and/or protect the naturallyoccurring pathways and processes of the body which occur in-vivo.

Accordingly, it is deemed likely that the primary function andcapabilities of the chosen biologically active molecule will differ andvary in many instances; and thus there are multiple purposes and a rangeof individual goals for the releasable substance, among which are thefollowing: (1) to serve as an antimicrobial agent—i.e., as ananti-bacterial or anti-fungal composition having a broad or narrowspectrum of activity; (2) to function as an anti-neoplastic compoundeffective against specific kinds of tumors; (3) to operate as aselective physiological aid—i.e., as a mediator which serves to avoidvascular complications such as blood coagulation or acts to prevent theformation of blood clots; and (4) to act as a pharmacologicalcomposition—i.e., as a drug or pharmaceutical which deactivates specifictypes of cells and/or functions to suppress or inhibit a variety ofdifferent humoral and cellular responses associated with or related toinflammation and the inflammatory response in-vivo. Examples of each arepresented hereinafter.

The Unique Electrospinning Perfusion Method of Manufacture

The Generation of Nanofibrous Tubular Structures

A preferred method for making the agent-releasing textile of the presentinvention is via the unique technique of electrospinning perfusion. Forthis purpose, an electrospinning perfusion assembly is erected whichcomprises, at a minimum, a rotating mandrel with a target surface whichcan be set at a pre-selected rotation speed; a needle fronted perfusioninstrument with a spinerette, such as a syringe, which can be set todeliver a liquid mixture at a pre-specified flow rate; an electricalcoupling for controlling and coordinating the electrical voltage appliedacross the perfusion needle and which is grounded to the rotatingmandrel; and a controllable supply of electrical power.

An admixture is prepared comprising a chosen non-biodegradable materialand a biologically active agent of choice. These components are blendedtogether into an organic liquid carrier. In one embodiment, the organicliquid carrier is cooled to an ice-cold (e.g. about 4° C.) temperature.For reasons that are not clear, this cooling step facilities the properformation of the admixture and speeds the dissolution of thenon-biodegradable material. For example, one preferred liquid admixtureor blending is obtained by combining 20% w:v polyethylene terephthalate(PET) with 1.5% w:v of an antimicrobial (e.g., Cipro or Diflucan), orwith 1.5% w:v of an anti-neoplastic compound (e.g., Paclitaxel,Everolimus, Sirolimus), in a sufficient quantity of ice-coldhexafluoroisopropanol (hereinafter “HFIP”). The resulting admixture issubsequently loaded into the electrospinning perfusion assembly.

For example, a 10 ml syringe with a stainless steel 18-gauge bluntspinneret (0.5 mm internal diameter) is then filled with the liquidpolymer blending and placed onto a Harvard Apparatus syringe pump forsubsequent perfusion. Perfusion is the action and the act of causing aliquid or other fluid to pass across the external surfaces of, or topermeate through, the substance of a tangible entity or a configuredphysical construct. Perfusion of a liquid or fluid thus includes thealternative actions of: a sprinkling, pouring, or diffusing through oroverlaying action; a covering, spreading, penetrating or saturatingaction (termed “suffusion”); a slow injection or other gradualintroduction of fluid into a configured space or sized internal volume(termed “infusion”); and a passage across a surface or through adiscrete surface or tangible thickness of matter, regardless of themechanism or manner of transfer employed for such fluid passage.

Once the admixture has been properly loaded, the electrical coupling andsyringe pump are activated and the admixture is electrospun onto thetarget surface. In one embodiment, the step of electrospinning iscarried out at a temperature which does not harm the biological activityof the biologically-active agent in the admixture. The reactiontemperature is, in one embodiment, ambient room temperature (20-25° C.),but when necessary or desired can be chosen to be within a temperaturereaction range of about 0-50° C.

Utilization of this assembly permits uniform coating of the liquidadmixture onto the surface of the mandrel; and the applied electricalvoltage can be varied as needed to control the formation of thenanofibers upon the mandrel's surface.

It will be recognized in particular that electrospinning over a broadrange of conditions Is possible for polyesters. Thus, a range ofdifferently formulated polyethylene terephthalates (or “PETs”) ofintrinsic viscosity [or “IV” as measured in OCP at 25° C.] that rangefrom less than 0.6 dl/g [IV] to greater than 1 dl/g [IV] can bedissolved in ice-cold 100% hexafluoroisopropanol. Electrospinningappropriately prepared HFIP solutions of such polyethyleneterephthalates results in the fabrication of nanofibrous textile fabricscapable of independent or combined release of diverse drugs, proteinsand genetic materials.

A Small Batch System

For fabricating small batches of product using this unique method, achemically resistant syringe with a stainless steel blunt spinneret canserve as a functional instrument for perfusion. Alternatively, ofcourse, any other tool, assembly or instrument capable of performingperfusion at a pre-selected flow rate and low reaction temperature canbe usefully employed.

In this small batch system, the perfusion syringe of the assembly isfilled with the prepared liquid mixture described above and placed ontoa Harvard Apparatus syringe pump. The perfusion rate is preferably setat 3 ml/hour at 25° C. If desired, however, the flow rate can beincreased and/or decreased to meet specific requirements. Similarly, thereaction temperature is preferably ambient room temperature (20-25° C.),but when necessary or desired can be chosen to be within a temperaturereaction range of about 0-50° C.

A PTFE-coated stainless steel mandrel (diameter ranges=0.75 mm-35 mm) ispreferably set at a jet gap distance of 15 cm from the tip of thesyringe needle. Gap distance can be varied at will to change the fiberdiameter size. The rotatable mandrel was then electrically grounded tothe power source, with the positive high potential source connected tothe syringe needle. The mandrel rotates or spins at a pre-selected rateof rotation throughout the act of liquid perfusion.

Perfusion

Perfusion of the polymer solution begins upon application of theelectric current to the tip of the syringe needle (typically 15 kV),which then moves at a preset constant speed and fixed distance from themandrel surface for a limited time period (typically about 40-90 minutesin duration). This process of manufacture is therefore termed“electrospinning perfusion”; and yields a fully fabricated, elongatednanofibrous textile conduit whose inner diameter size corresponds to theoverall diameter of the mandrel (in this instance, 4 mm).

When using a single nozzle (or syringe needle), it was that increasingelectrospinning time significantly beyond about 40 minutes increased therigidity of the resulting nPET material. However, multiple nozzles (orsyringe needles) can be used concurrently to reduce the time required tofabricate tubular structures of the appropriate rigidity. The use ofmultiple injection streams to Increase production rates is a familiarconcept to those skilled in the art; and, accordingly, the use ofmultiple nozzles lies within the scope of the present invention.

Optional Follow-Up Processing

When the process is used to make certain kinds of medical articles suchas synthetic vascular graft prostheses, a crimping procedure is employedas an optional, but very desirable, follow-up process. Accordingly,after being formed as a hollow tube by electrospinning perfusion, thethickness and girth of the originally formed fibrous composite wall andexterior surface preferably is then intentionally altered into a crimpedstructural form via a limited heat (low temperature) set technique,followed by compression of the fibrous composite wall, in order toprovide kink-resistance for the elongated tube.

In brief, the end portions of the formed hollow tube (appearing about 1cm from each end of the mandrel) are cut off and discarded. Theremainder of the elongated hollow tube is then stretched 25% of thestarting segment size while on the mandrel in order to provide a setstrain across the fibers, a manipulation that occurs in normal fiberextrusion. The stretched tubes are then immediately exposed to 100%ethanol for 2 hours time at room temperature (or in 100% ethanol for 30minutes with sonication) in order to remove the residual solvent,followed by air-drying overnight at room temperature. This crimpingtechnique permits a user to form specific shapes (e.g. bends, etc) inthe fabric without using high-temperature melt techniques which woulddamage the biologically-active agent.

The Generation of Flat Sheet Nanofibrous Textile Fabrics

Similar in its essentials to the technique described above. DACRON chipswere dissolved in ice-cold 100% hexafluoroisopropanol (19% w:v) andmixed on an inversion mixer for 48 hours in order completelysolubilize/e the chips. The self-contained, semi-automatedelectrospinning apparatus containing a Glassman power supply, a HarvardApparatus syringe pump, an elevated holding rack, a modifiedpolyethylene chamber, a spray head with power attachment and areciprocating system was again used.

The stirrer was used to provide a holding chamber for the new flatcollecting plate employed to generate a sheet format. The design of thissurface is based upon the collecting plate employed by Li et. al. [seeLi W J, Laurencin C T, Caterson E J, Tuan R S, Ko F K., “Electrospunnanofibrous structure: A novel scaffold for tissue engineering”. JBiomed Mater Res 60:613 (2002)]. In short, a flat 12 cm.times.10 cmcopper plate, containing a 6 cm stainless steel rod extending from theunderside of the plate was designed and grounded to the power source.

A 10 ml chemical-resistant syringe was filled with the polymer liquid. Astainless steel 18-gauge blunt spinneret (0.5 mm internal diameter) wasthen cut in half, with the syringe fitting end connected to thepolymer-filled syringe. Nalgene PVC tubing was connected to the syringefilled with the polymer solution followed by connection to the otherhalf of the blunt spinneret within the spray head. The line was thenpurged of air, with the syringe then placed onto the syringe pump. Thehigh potential source was connected to the spray head tip, with theplate set at a jet gap distance of 15 cm from the tip of the needle. Theperfusion rate was set at 3 ml/hour at 25° C.

Perfusion of the polymer liquid was started upon application of thecurrent to the tip of the needle (15 kV) with electrospinning proceedingfor 1 hour and 40 minutes, with rotation of the plate 20 degrees every20 minutes. This resulted in a flat, planar sheet of nanofibrous textilematerial being formed.

The agent releasable nanofibrous textile formed by the electrospinningmethod described above has a number of unique structural features whichare the direct result and characteristic of its unique mode and mannerof manufacture.

1. The agent-releasing textile fabricated via one of the two differentelectrospinning perfusion techniques will yield a discrete tubulararticle of fixed inner-wall and outer wall diameters, and a solid wallgirth and configuration formed of a nanofibrous composite composition.The material substance of the fabricated wall typically shows that thesynthetic substance is present as discrete fibers about 10⁻⁸ meters indiameter size. The fiber size is clearly demonstrated by the empiricaldata presented subsequently herein.

2. The interior wall surface and the exterior wall surface of thetubular structure comprising the agent-releasing textile are markedlydifferent owing to the crimping and heat setting treatments followingthe initial electrospinning perfusion steps of the methodology. Thus,the exterior wall surface can possess a crimped and a somewhat irregularappearance. In comparison, the interior wall surface and the internallumen of the conduit as a whole presents a smooth, regular, and evenappearance which is devoid of perceptible projections, lumps,indentations, and, roughness.

3. The nanofibrous composite material substance of the textile fabric,whether existing in tubular structure form or in planar sheet form, isresilient and can be prepared in advance to provide varying degrees offlexibility, springiness, suppleness, and elasticity. Moreover, thenanofibrous biocomposite wall is durable and strong; is hard to tear,cut, or breakup; and is hard-wearing and serviceable for many years'duration.

4. The nanofibrous material substance of the agent releasable textile,whether present in tubular structure form or in planar sheet form, isbiocompatible with the cells, tissues and organs of a living subject;and can be implanted surgically in-vivo without initialing or inducing amajor immune response by the living host recipient. While asepticsurgical technique and proper care against casual infection during andafter surgery must be exercised, the agent releasable textile can beusefully employed for a variety of applications in-vivo.

The Major Benefits and Advantages of the Electrospinning PerfusionTechniques

The electrospinning perfusion technique-whether employed to fabricatetubular structures or flat sheets, has a number of advantages overconventionally known manufacturing processes. These include thefollowing:

A first benefit is dial no exogenous binders, cross-linking compounds,or functional agents are required by the process either to form thesubstance of the fabric or to maintain the integrity of the fabricatedtextile. The synthetic substance prepared in liquid organic solvent canbe generated directly into nanofibrous fabric form via the low reactiontemperatures (typically ranging between 0-50° C.) permitted and used bythe electrospinning perfusion process. In addition, the nanofibers ofthe fabric act to seal the interstices of the composite material;therefore, no sealants as such are required. This manufacturingtechnique also benefits the manufacturer in that the technology is not adipping or immersion method of preparation, which can be awkward anddifficult to perform; or is a process which typically requires theaddition of heat, such as if a conventional melt spinning method offiber formation were employed.

A second benefit is that the electrospinning perfusion technique yieldsa textile fabric formed as a nanofibrous composite in which the fibers(e.g., PET) exist independently and are visibly evident throughout thematerial of the textile. This structural distribution of discrete fiberswithin the fabric adds strength and flexibility to the textile as awhole. Also, the presence of these fibers collectively provides sitesinto which diverse biological agents (such as antimicrobials,anti-neoplastic agents, and the like) can be temporarily incorporatedand indefinitely, although non-permanently, immobilized until such timeas the textile takes up fluid—i.e., any aqueous and/or organic liquid.

A third benefit is the capability for direct incorporation ofbiologically-active agents onto the nanofibrous material, whatever itsfinal shape and structure. This process holds several key advantagesover other conventionally known methodologies in that:

The active agent is incorporated into the fabricated nanofibrousmaterial without molecular modification, and is non-permanentlyimmobilized within each individual fiber surface as the individualfibers are formed.

No one particular mechanism of incorporation is responsible for theactive agent becoming non-permanently immobilized within each individualfiber of the fabricated nanofibrous material; and thus any and all ofthe commonly known mechanisms—such as absorption, adsorption, polarity,ion attraction, and the like—may be involved.

The amount of active agent can be adjusted within the bulk polymerdepending on the specific or intended application.

No cross linking agents are needed, or used, or desired at all, therebyavoiding concerns over drug carrier toxicity, biocompatibility, andmutagenicity.

Low reaction temperatures are used during the fiber/fabric formationprocedure, thus maintaining the biologic activity of the active agent.

Active agent elution from the textile fabric is controlled and sustainedover time, as shown in the experimental studies and empirical datapresented hereinafter.

The Releasable Anti-Neoplastic/Anti-Prolerative Agents

Paclitaxel, also known as Taxol, a diterpenoid-structured molecule shownby FIG. 3, is a potent anti-neoplastic agent. Paclitaxel has been shownto inhibit vascular smooth muscle cell (VSMC) proliferation, migrationand inflammation. Additionally, Paclitaxel has been shown to inhibit thesecretion of extracellular matrix by VSMCs, a major component ofneointima formation leading to vessel restenosis. Paclitaxel stabilizesand enhances assembly of polymerized microtubules, an importantcomponent of the cytoskeleton involved in cell division, cell motilityand cell shape. Other examples of anti-proliferative/anti-neoplasticagents such as Sirolimus, Everolimus, Tacrolimus, 5-FU, daunomycin,mitomycin and dexamethasone can also be used.

Additionally, microtubules are involved in signal transduction,intracellular transport and gene activation. Paclitaxel has shownpromise as a treatment for various types of cancers as well as for theprevention of restenosis following stent placement.

Nevertheless, when Paclitaxel is incorporated into a hydrophobic carrierpolymer coated onto a metallic stent, it elutes for only 10-14 days.Other research groups have attempted to incorporate Paclitaxel intobiodegradable polymers that would comprise the stent. However.Paclitaxel activity was significantly reduced due to the melt extrusionprocess for the fibers.

This issue would not be a problem with the present invention due to thelow temperature formation of the nanofibrous polyethylene terephthalate(PET) fibers. Therefore, the fabrication of a nanofibrous polyethyleneterephthalate (PET) material with a slow-releasing anti-neoplastic agentsuch as Paclitaxel would be particularly effective and medicallyapplicable to endovascular stents and prosthetic vascular grafts, bothof which currently experience neointimal hyperplasia. Additionalexamples of other active anti-neoplastic agents suitable for use in thepresent invention include Rapamycin and Dexamethasone.

The Fluoroquinolone Antibiotics

Antibiotics vary in structural type, spectrum of activity, and clinicalusefulness. Fluoroquinolones such as Ciprofloxacin (hereinafter “Cipro”)are shown structurally by FIG. 1, and are of particular use and value inthis invention. Quinolone antibiotics are chemically stable, andeffective at tow concentrations against the common clinicallyencountered organisms, particularly those bacteria responsible forbiomaterial infection. These antibiotics also have structural features(solubility, molecular mass, and functional groups) that coincide withthose of textile dyes known to have interactions with polyethyleneterephthalares.

This family of antibiotics has expanded considerably—Ciprofloxacin,Ofloxacin, Norfloxacin, Sparfloxacin, Tomafloxacin, Enofloxacin,Lovafloxacin, Lomefloxacin, Pefloxacin, Fleroxacin, Avefloxin,Levofloxavin, Moxifloxacin and DU6859a; and the fluoroquinolone familyas a whole has become the drug of choice for many applications. Theseantibiotics are effective at low concentrations; and hold an idealantimicrobial spectrum against microorganisms most commonly encounteredclinically in wound infection, with significant activity against manyrelevant pathogens-such as S. aureus, methicillin-resistant S. aureus,S. epidermidis, Pseudomonas species, and Escherichia coli. Moreover,Fluoroquinolones are heat stable; are of 300-400 r.m.m.; and have manystructural features analogous to dyes. Accordingly, this family ofantibiotics possesses those characteristics which are highly desired foruse with the present invention.

A list of some representative antimicrobial/antiseptic agents that canbe used solely or in conjunction with the fluoroquinolones is includesβ-lactams, biguanides cephalosporins, chloramphenicol, macrolides,aminoglycosides, quaternary ammonium salts, tetracyclines,sulfur-containing antimicrobials, silver-containing compounds,bis-phenols (triclosan), vancomycin, novobiocin and steroids (fusidicacid)

The Anti-Fungal Agents

Development of antifungal agents has been on the rise over the past twodecades due to a significant increase of superficial (i.e. nail beds)and invasive (i.e. blood-borne and medical-device related) infections.Fluconazole, known as Diflucan, a triazole-structured antifungal agentintroduced in early 1990 and structurally shown by FIG. 2, has emergedas one of the primary treatments for Candida infections. The mode ofaction of Diflucan is the inhibition of 14.alpha.-lanosterol demethylasein the ergosterol biosynthetic pathway, and results in the accumulationof lanosterol and toxic 14.alpha.-methylated sterols in the fungalmembrane. Similar to the selection of Cipro. Diflucan has structuralfeatures (solubility, molecular mass, and functional groups) thatcoincide with those of textile dyes known to have interactions withpolyethylene terephthalate fibers. A agent-releasing textile combiningpolyethylene terephthalate with a slow-releasing antifungal agent suchas Diflucan will have a marked impact on topical and implantablebiomaterials such as medicated pads (useful for nail bed and skininfections), tampons (using localized release for yeast infection) andcatheter cuffs.

Other examples of anti-fungal agents typically will include amphotericinB, Nystatin, Terbinafine, Voriconazole, Echinocandin B and Itraconazole

The Antimicrobial Peptides

A novel class of antimicrobial agents known as antimicrobial peptides(or “AMPs”) has been discovered during the past two decades. These“natural” antimicrobial agents, which consist of a large number of lowmolecular weight compounds, have been discovered in plants, insects,fish and mammals, including humans [see for example. Marshall S H &Arenas G., “Antimicrobial peptides: A natural alternative to chemicalantibiotics and a potential for applied biotechnology”, J Biotech 6(2):1(2003)]. These peptides, whose composition can range front 6-50 aminoacids, have been shown to have an important role in innate immunity.There are 5 general classifications for AMPs [see for example, SarmafilkA., “Antimicrobial peptides: A potential therapeutic alternative for thetreatment of fish diseases”, Turk J Biol 26:201(2002)], which are basedon the three-dimensional structure of the peptide as well as thebiochemical characteristics. These groups consist of: (1) linearpeptides without cysteine residues or hinge region; (2) linear peptideswithout cysteine residues and a high proportion of certain amino acids;(3) antimicrobial peptides with one disulfite bonds that form a loopstructure; (4) antimicrobial peptides with two or more disulfite bonds;and (5) antimicrobial peptides that have been derived from other largerproteins via post-translational processing.

AMPs have shown broad spectrum antimicrobial activity against bothgram-positive (i.e., Staphylococcus aureus and epidermidis) and negative(i.e., Pseudomonas aeruginosa, E. coli) bacteria. Some AMPs have alsobeen shown to be effective against fungus [see for example. De Lucca A.J., “Antifungal peptides: Potential candidates for the treatment offungal infections”, Expert Op Invest Drugs 9(2):273 (2000); andSelitrennikoff C P, “Antifungal proteins”, Appl Environ Microbiol67(7):2883 (2001) and several antibiotic-resistant bacteria such asMycobacterium tuberculosis [see for example, Linde C M A, Hoffier S E,Refai E, Andersson M., “In vitro activity of PR-39, aproline-arginine-rich peptide, against susceptible and multi-drugresistant Mycobacterium tuberculosis”, J Antimicrob Chemother 47:575(2001); Miyakawa Y, Ratnakar P, Rao A G, Costello M L, Mathieu-CostelloO, Lehrer R I, Catanzaro, A., “In vitro activity of the antimicrobialpeptides human and rabbit defensins and porcine leukocyte protegrinagainst Mycobacterium tuberculosis”. Infect Immun 64(3):926 (1996); andSharma S, Verma I, Khuller G K, “Therapeutic potential of humanneutrophil peptide 1 against experimental tuberculosis”, AntimicrobAgents Chemother 45(2):639 (2001)].

Although the mode of action by these peptides has not been fullyelucidated, it is postulated that many of these peptides interactdirectly with the bacteria wall, creating small channels (pores) whichcauses membrane destabilization, thereby depleting the bacteria of itscytoplasmic content [see for example, Matsuzaki K., “Why and howpeptide-lipid interaction utilized for self defense? Magainins andtachyplesins as archetypes”, Biochemica Biophys Acta 1462(1-2):456(1999)]. While effective against bacteria walls, there appears to belimited affinity for eukaryotic cells possibly due to the differentcomposition and net charge of the membranes. Several AMPs (i.e., Nisinand Daptomycin) have been recently approved by the FDA for commercialand medical markets. This acceptance paves the way for utilizing otherAMPs such as pleurocidin. Additionally, federal standard testingprocedures, which were used to provide safety and efficacy data forthese AMPs, have been established. Other representative types of AMPsinclude Cationic peptides such that Cecropins, Defensins, Thionins,Amino Acid-Enriched Histone-Derived Beta-Hairpin and other Natural andFunctional Proteins. Further examples of anionic peptides includeAspartic Acid-Rich, Aromatic Dipeptides and Oxygen-Binding Proteins.

The Analgesic Agents

Analgesic agents are widely used in human and veterinary medicine inorder to prevent inflammation, thereby reducing pain and other symptomssuch as itching and swelling. These agents have structural propertiesthat are comparable to standard textile dyes such as molecular weight,functional groups and benzene-ring based composition. Exemplifying suchanalgesic agents are Diphenhydramine Hydrochloride. Meloxicam,Hydrocortisone Acetate, Pramoxine Hydrochloride, Lidocaine andBenzocaine.

The Anti-Viral Agents

Antiviral agents have been used to combat viral infections ranging fromthe flu to HIV infection and organ transplant rejection. Examples ofsome antiviral agents include Oseltamivir (Flu), Zanamivir (Flu),Saquinavir (HIV), Ritonavir (HIV), Interferon (HIV/Implant Rejection).

Other Classes of Suitable Biologically Active Agents

A number of other classes of biologically active agents can also be usedin the agent releasable textile. All of these choices are biochemicalmediators which can be initially immobilized via the electrospinningtechnique without serious deterioration, and then subsequently releasedfrom the nanofibrous textile fabric upon uptake of water. Representativeexamples of such classes comprising additional suitable biologicallyactive agents are presented by Tables 9, 10, and 11 of U.S. Publicationno. 2006/0200232A1, the content of which is incorporated by reference.

The Medical Articles Fashioned from the Agent Releasable Textile

It is expected and envisioned that each agent-releasing textile can beemployed in the alternative either (1) as a configured tubular conduitwhose internal lumen is usefully employed for the conveyance of fluidsin-situ; or (2) as a solid mass of flat or planar nanofibrous sheetfabric which achieves its intended purpose without regard to or actualuse of any internal lumen within the textile fabric. Some representativeexamples of the tubular format include vascular articles such asarterial vascular grafts; venous vascular grafts; prostheses foraneurysms; liners and covers for stents (coronary or endovascular) aswell as non-vascular devices including catheter cuffs and coating forwires for transdermal devices (pacemaker leads). Illustrative examplesof flat sheet formats include wound dressings such as treatmentdressings, films, and/or sheets; gauze pads; absorbent sponges;bandages; and sewing cuffs. Further examples include transdermal releasepatches such as infection treatment; skin tumor treatments; andfinger/toenail treatment. Further examples include personal hygieneproducts such as tampons; and contraceptive delivery.

Some Intended Clinical/Therapeutic Applications for the Invention

The kinds of clinical/therapeutic applications for the prepared medicalarticles and devices are intended to include major traumatic woundscaused by accident, negligence, or battlefield conditions; plannedsurgical incisions and invasive body surgical procedures performed underaseptic conditions; transcutaneous incisions and vascular openings forcatheter insertion and blood vessel catheterization procedures; andother body penetrations and openings made for therapeutic and/orprophylactic purposes.

The medical articles provided by the present invention thus are intendedand expected to be manufactured as pre-packaged and pre-sterilizedtextile fabric articles; be an item which can be prepared in advance, bestocked in multiples, and be stored indefinitely in a dry state withoutmeaningful loss of biological function or efficacy; and serveeffectively in the treatment of disease, disorders, and pathologicalconditions under many different clinical circumstances.

The medical articles should be manufactured and tailored in advance tomeet a wide range of intended use circumstances or contingenciesexpected to be encountered in a particular situation. For this reason,the constructed textile article can and should alternatively be preparedas a thick cloth and as a thin gauze; as a solid-walled configured tube;and as a delicate film. Equally important, the resulting construct maytake physical form either as a stiff, inflexible and unyielding mass oras a very flexible and supple layer; have a varied set of dimensions andgirth; appear as both a geometrically symmetrical or asymmetricalconfigured fabric; and can exist even as a slender cord or string-likelength of material.

Medically, the agent releasable textile articles of the presentinvention can be employed in-vivo in the following ways: topically orsubtopically; transcutaneously, percutaneously, or subcutaneously; orinternally within the body's interior; viscerally or Immorally; andapplied to any kind of body cavity, body tissue or body organ withoutregard to anatomic site or location.

Experiments, Empirical Data, and Results

To demonstrate the merits and value of the present invention, a seriesof planned experiments and empirical data are presented below. It willbe expressly understood, however, that the experiments described hereinand the results provided below are merely the best evidence of thesubject matter as a whole which is the present invention; and that theempirical data, while limited in content, is only illustrative of thescope of the present invention as envisioned and claimed.

An illustrative recitation and representative example of the presentinvention is the preferred manner and mode for practicing themethodology is also presented below as part of the experimental method.It will be expressly understood, however, that the recited steps andmanipulations presented below are subject to major variances and markedchanges in the procedural details; all of which are deemed to be routineand conventional in this field and may be altered at will to accommodatethe needs or conveniences of the practitioner.

Commercial Applications

Medium and Small-Diameter Artificial Arteries

Problem: Peripheral arterial disease (PAD) is estimated to affect some8-10 million people in the United States alone. For patients needingsurgical intervention, there is currently no prosthetic vascular graftaccepted for vascular reconstruction in the lower extremities. Thenumber of patients requiring this procedure is projected tosignificantly grow over the next decade due to an aging population inconjunction with the earlier prevalence of diabetes and hypertension asa result of increasing obesity rates in the general population. Anautologous vessel graft is the first and currently only accepted choicein most arterial grafting procedures for these anatomic areas. However,this situation becomes problematic when disease progression has occurredthroughout the vasculature or when the patient has utilized all of theharvestable veins for other surgical procedures, thereby leaving noviable arterial graft alternative for the patient. These complicationsresult in significant morbidity and mortality rates. The two mostcommonly used synthetic materials, polyethylene terephthalate (polyesteror PET) and expanded polytetrafluoroethylene, have been used extensivelyover the last several decades for medium and large diameter grafts, buthave failed when evaluated for use as small-diameter (<5 mm internaldiameter) vascular prostheses. Various technologies have been developedto improve biocompatibility of prosthetic grafts. While several of thesetechnologies have shown early promise, none have been fully-accepted asa viable alternative to autologous vessel for distal bypass surgery.

Composition to address problem: a copolymer polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT))and bioactive agents: Anticoagulant (recombinant hirudin or Argatroban),antiproliferative (paclitaxel, everolimus, sodium butyrate and/orsilencing siRNA), growth promoting (vascular endothelial growth factor,fibroblast growth factor) and/or antimicrobials (antibiotics,antimicrobial peptides, naturally-occurring antimicrobial proteins). Theshape is a straight tubular construct or tapered internal diameter; canalso incorporate crimp or inner wall reinforcement to provide greaterflexibility. Dimensions: 0.75 mm internal diameter and larger (>40 mm);length from 1 cm-60 cm.

Synthesis Procedure: A PET/PBT (17.5%/2% w:v) polymer solution (15 ml)was prepared in ice-cold HFIP and mixed for 48 hours. The PET/PBTpolymer solution was then equally divided (5 ml/vial). One portion ofthe polymer solution was left unmodified, serving as the non-drug loadedpolymer solution (control). One of each of the bioactive agents from theselected categories was added to the other respective polymer solutionsafter 3 days of mixing. These solutions were mixed for two additionaldays before electrospinning. These polymer solutions (control anddrug-loaded) were each added to individual 5 ml syringes and electrospunseparately for 45 minutes onto either a 4 mm diameter Teflon-coatedstainless steel mandrel or tapered steel mandrel (6 mm-4 mm). Jet gapdistance was set at 15 cm. Perfusion of the polymer (3 ml/hour) wasinitiated upon voltage application (+20 kV). Electrospun materials werecut in a longitudinal direction to form flat sheets. The electrospuntubular constructs (control and drug-loaded nPBT-PBT) were removed offof the mandrel, stretched 25% of the original length and post-treated toremove any residual solvent by sonication in 100% ethanol for 30 minutesfollowed by then sonication in distilled water for 2 minutes. Graftswere air-dried overnight at room temperature. Electrospun materials weresterilized via ethylene oxide (EtO) using an Anprolene Sterilizer (230%RH, 12 hour cycle).

Hemodialysis Access Graft

Problem: End-Stage Renal Disease (ESRD) is a disease affecting more thana half million Americans. Current gold standards for hemodialysisaccess, radial cephalic vein fistulas and autogenous saphenous veins,have significant problems associated with their use. Arteriovenousfistulas (AVFs) need a long time to heal before access (6 weeks to 6months). Surgical time for autologous grafts is significantly increasedas a result of harvesting the vein as well as treatment prior toimplantation. Additionally, most patients do not have veins to utilizedue to co-morbidity, prior harvesting for a surgery or the need to savethe vessel for a different surgical procedure (e.g. coronary artery ordistal bypass). Viable veins may also not be available due to diseaseprogression. Lastly, primary patency rates for these autogenous graftsafter two years of implantation is approximately 20%, althoughcumulative patency rates as high as 89% have been reported aftersurgical/pharmacological intervention.

Synthetic grafts made of ePTFE are the current standards for syntheticvascular access grafts. These grafts have (depending on the study)comparable or worse primary patency rates than autogenous grafts and,similar to autogenous grafts, take a significant time to heal (at least2-4 weeks) thereby preventing instant hemodialysis access. Theseprosthetic alternatives are also relatively stiff compared to the nativevessels and have issues related to infection. The other issuesassociated with ePTFE grafts are seroma formation and occlusion due tointimal hyperplasia. Various efforts to improve the patency throughcoatings (carbon coating), impregnations (fibroblast growth factors) andsurface protein binding (heparin) have failed to improve overalllong-term patency rates. To date, thrombosis, infection and the lack ofearly access via needle puncture are the biggest issues associated withePTFE grafts. Grafts made of a composite material (Vectra™; Thoratec LabCo., Pleasanton, Calif., USA) comprised of polyurethane, silicone andPET fibers (as reinforcement) have recently entered the market. Thegraft has the self-sealing property and healing behavior comparable toePTFE grafts. Additionally. Vectra™ does not require a long healing timeprior to the first puncture. However, the solid silicone film locatedwithin two layers of polyurethane in order to impart impermeability andself-sealing to the graft prevents complete healing of the graft. Thehigh elasticity of the graft also causes kinking of the native veinresulting in stenosis.

Composition to address problem: a copolymer of polyethyleneterephthalate (PET) and polyurethane (PU) and bioactive Agents:Anticoagulant (recombinant hirudin. Argatroban or Bivalirudin),antiproliferative (paclitaxel, everolimus, sodium bulyrate and/orsilencing siRNA) and antimicrobials (antibiotics, antimicrobialpeptides, naturally-occurring antimicrobial proteins) Shape: Straighttubular construct; incorporates crimp within mandrel to provide greaterflexibility. Dimensions: 6-8 mm internal diameter and smaller, lengthfrom 20-80 cm.

Synthesis Procedure: A polymer solution (10% w:v, 30/70 PET/PU) inconjunction with antibiotic (Moxifloxacin), anticoagulant (recombinanthirudin or rHir) and anti-proliferative (Paclitaxel or Pac) (1.5%, 1%and 1% w:v, respectively) was prepared. This solution was diluted anadditional 20% with HFIP and mixed for an additional 2 hours. FurtherHFIP dilution of the original polymer solution prevented anycomplications during graft synthesis while improving final electrospunmaterial. This diluted solution was then electrospun onto Teflon-coatedstainless steel mandrels with a spring loaded within the mandrel tocreate the crimped structure (40 cm length; 6.2 mm diameter), resultingin a graft with an internal diameter of 6 mm and a length of 25 cm. Thegraft was then post-treated to remove any residual solvent by sonicationin 100% ethanol for 30 minutes followed by sonication in distilled waterfor 2 minutes. Grafts were then air-dried overnight at room temperaturefor 72 hours. BioAccess grafts were ethylene-oxide (EtO) sterilizedusing an Anprolene Sterilizer at BioSurfaces, Inc. (cycle time=12 hours,room temperature, humidified conditions).

Ventricular Assist Device Tubular Device

Problem: Heart failure affects over 4.7 million Americans, with 550,000new cases diagnosed each year. Of these cases, approximately 50,000 to100,000 patients are in late-stage heart failure with only 8% of thesepatients surviving two years without undergoing a heart transplant orimplantation of a ventricular assist device (VAD). Although VADs haveimproved the quality of life for patients in late-stage heart failure,only 2,000 patients receive VADs each year due to the high morbidity-and mortality associated with these devices. VADs, similar to allmedical devices implanted within the vasculature, are prone to two majorcomplications: thrombosis/thromboembolic phenomenon and infection. Theannual healthcare cost for this major disorder is estimated at $10 to$40 billion. Significantly reducing these adverse complications wouldshift VAD use from “bridge to transplant” to “destination therapy”,increasing the potential market from the current $100 million annuallyto $2.5 billion. Development of this technology may also haveapplication for other implantable devices such as hemodialysis accessgrafts as well as medium-bore prosthetic arterial grafts and sewingcuffs comprised of polyester, in which thrombosis and infection areassociated with their use.

Composition to address problem: a copolymer polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT))and Bioactive Agents: Anticoagulant (recombinant hirudin, Argatroban orBivalirudin) and antimicrobials (antibiotics, antimicrobial peptides,naturally-occurring antimicrobial proteins). Shape: Straight tubularconstruct; can also incorporate crimp or inner wall reinforcement toprovide greater flexibility. Dimensions: 4 mm internal diameter andlarger; length from 5 cm-60 cm.

Synthesis Procedure: Polyester (17.5% w:v PET and 2% w:v poly(butyleneterephthalate) or PBT) polymer solutions were prepared in ice-cold 100%hexafluoroisopropanol (HFIP). This polymer solution was mixed on aninversion mixer for 48 hours in order completely solubilize bothcomponents. To this solution, rHir (1% w:v) and Cipro (1.5% w:v) wasadded and mixed an additional two days. The self-contained,semi-automated electrospinning apparatus described in the contract wasemployed. Utilization of this system permitted uniform coating of thepolymer onto the PTFE-coated stainless steel mandrel (diameter=6 mm).The high potential source was connected to the spray head tip. Themandrel, set at a jet gap distance of 15 cm from the tip of the needle,was then grounded to the power source. The perfusion rate was set at 3ml per hour at 25° C., with perfusion of the polymer started uponapplication of the current to the tip of the needle (+15 kV).Electrospinning time was increased from 60 minutes to 90 minutes inorder to significantly increase wall thickness. After electrospinning,the end portions of the conduit (1 cm from each end of the mandrel) werecut off and discarded. The remaining graft was stretched 25% of thestarting segment size while on the mandrel. Conduits were left on themandrel and placed into 100% ethanol and sonicated for 30 minutes,followed by a sonication in sterile distilled water for 2 minutes inorder to remove residual HFIP solvent. These BioSpun-VAD conduits werecut to length and ethylene oxide (EtO)-sterilized.

Sewing Cuff Ring (Heart Valve Repair and Artificial Heart ValveAttachment)

Problem: Cardiac valve repair or replacement is indicated whenprogression of degenerative disease or bacterial infection of the nativevalve results in valvular dysfunction, thereby impacting cardiac outputBoth procedures require the use of a woven or knitted polyester ringwith an internal reinforcement (Teflon, silicone or metal) to eitherstabilize the native valve (annuloplasty ring) or to attach a prostheticheart valve (sewing cuff). Bacterial infection (prosthetic valveendocarditis or PVE) is a major complication associated withimplantation of these devices. Infection of these devices can emerge viatwo mechanisms. Nosocomial infection at the time of surgery orpost-operatively occurs approximately 1-4% of all valves implanted,resulting in significant morbidity and mortality. Blood streaminfections (bacteremia) seeded at the implantation site prior to surgeryhave been shown to also occur in approximately 33% of all PVE cases,with a mortality rate above 50% from this serious complication.Staphylococcus aureus (S, aureus) and epidermidis (S. epidermidis) aswell as Streptococci are shown to be responsible for 25-50% of all valveinfections. Perioperative parental antibiotics often fail to permeatethe avascular spaces immediately around the biomaterial once pathogenshave adhered. The health care cost associated with treating PVE isprojected to be greater than $60,000 per patient, with the annual marketfor cardiac surgery devices projected to range from $700 million to $1.4billion.

Overall, valvular disease affects 2.5% of the United States population(this percentage is higher in older age groups). Over 90,000 mechanicaland bioprosthetic valves are implanted in the United States each year,with over 280,000 valves implanted worldwide. While the emergence oftranscatheter heart valve therapy will reduce selection of these devicesfor certain procedures, overall valve use is still projected to increasedue to an aging population and, to a lesser extent, a more aggressivesurgical approach to mitral valve insufficiency. Additionally, higherincidences of obesity and diabetes are expected to increase thesenumbers drastically. Currently, there are no clinically availableinfection-resistant prosthetic valves or sewing cuffs/annuloplastyrings. Due to the inertness of prosthetic valves, these annuloplastyrings and sewing cuffs are logical targets to provide localizedantimicrobial delivery.

Composition to address problem: Polymer: Combination of polyethyleneterephthalate (PET) and polyurethane (PU), Bioactive Agents:Anticoagulant (recombinant hirudin, Argatroban or Bivalirudin) andantimicrobials (antibiotics, antimicrobial peptides, naturally-occurringantimicrobial proteins). Shape: Ring shaped device, thickness can bevaried. Dimensions: 5-35 mm internal diameter.

Synthesis Procedure: The electrospun sewing cuff is a compositenanofibrous construct, involving electrospinning of two polymersolutions. For both control and drug-loaded sewing cuffs (BioCuffs), a10% (w:v) PU polymer solution (Chronoflex C Polycarbonate Polyurethane;80A Durometer) was prepared in ice-cold 100% HFIP. Another polymersolution of 17.5% (w:v) PET and 2% (w:v) PBT was also prepared inice-cold 100% HFIP. Each solution was then mixed for 48 hours on aninversion mixer. These two solutions were then electrospun sequentiallyto form the nanofibrous control sewing cuff material. To synthesizedrug-loaded sewing cuffs (BioCuffs), after mixing the polymer solutionsfor 48 hours, the volumes of both solutions were diluted by adding 50%more HFIP. Cipro (1.5% w:v) was then added to the diluted PET-PBT and PUsolutions and both polymer solutions were mixed for an additional 24hours before electrospinning. A gap distance of 15 cm was set from theneedle port to the collecting surface, which was a 9.5 mm diameterzinc-plated steel mandrel with a roughened surface. This mandrel wasrotated at a constant speed of 270 rpm. Perfusion of the PET-PBT polymer(3 ml/hour) was started upon application of the current to the needle(+20 kV), with electrospinning proceeding for 5 minutes in the case ofthe control BioCuffs, or for 7.5 minutes in the case of the drug-loadedBioCuffs (1.5× duration for diluted polymer solution). Immediately afterelectrospinning the PBT-PBT layer, the Teflon tubing was connected tothe PU-filled syringe and purged of residual polymer solution, withelectrospinning of the PU layer proceeding for 15 minutes in the case ofthe control BioCuffs, or for 22.5 minutes in the case of the drug-loadedBioCuffs. After electrospinning both layers, the coated rods were washedin ethanol for 30 minutes with sonication, followed by a 2 minutesonication in distilled water to remove all traces of residual solvent.The edge of the material was rolled towards the opposite end of the rod,while measuring the thickness of the BioCuff with calipers whenapproaching the desired thickness of the final product. The material wasthen cut at the edge of the rolled sewing cuff. The detached cuff wasrolled off the remainder of the rod length and the edge fused tocomplete cuff formation. Cuffs were air-dried at room temperature.Sewing cuffs, with and without drugs, were then sterilized by ethyleneoxide (EtO) via an Anprolene Sterilizer (25° C., 10 psi, cycle time=12hours). BioCuffs were then evaluated for surface fluorescence (Cipro)via a hand-held UV light and compared to control sewing cuffs.

Nanofibrous Bioactive Suture Materials

Cardiovascular disease (CVD) is the leading cause of death in the US,constituting over $272 billion of all national health expenditures. Mostsequelae of CVD are confined to peripheral or coronary arteries, asblood vessels become occluded and require either surgical bypassprocedures or percutaneous intervention (PCI) to restore blood flow tovital organs and limbs. As rates of obesity and diabetes climb to recordlevels each year, the prevalence of CVD continues to increase. By 2030,41% of Americans will likely have some form of CVD. While PCI and drugeluting stents (DES) have become popular, recent studies show thatbypass holds many advantages over PCI in terms of cost (due to the needfor repeat PCI procedures), life expectancy, and quality of life forpatients expected to live more than 2 years. The vast majority of recentstudies comparing bypass grafts to stents corroborate this. Yet, bypassgrafts still suffer early and late failure due to intimal hyperplasia(IH), the chronic excessive proliferation of smooth muscle cells (SMCs)as a response to injury of the blood vessel. In bypass grafts, IH occursprimarily at the anastomosis, where the suture joins the vein to theartery. Blood flow through the vein graft is gradually constricted bySMC overgrowth at the anastomosis until the vessel becomes occluded bythrombosis, causing 39% of bypass grafts to fail within 10 years and 50%to fail by 15 years. Harvesting and denuding are also known to cause ahyperproliferative response due to injury, but this response is acuteand short-lived, and eventually regresses. IH occurs to a lesser degreeat the floor of the native artery as well, where rerouted blood altersthe natural hemodynamics of the vessel. However, this IH is anadaptation to a hemodynamic stagnation zone, and is therebyself-limiting once thickening at the floor of the artery incurs naturalhemodynamic flow conditions. Alternatively, the presence of commerciallyused sutures generates a persistent, chronic hyperproliferative responsethat is the leading cause of failure in bypass grafts. Thus, controllingIH at anastomoses is our prime objective.

While antiproliferative DBS have improved revascularization rates by 55%compared to bare metal stents, sutures used for bypass grafts are yet 10incorporate an antiproliferative drug eluting strategy. This is a majorgap in the state of the art for vascular surgery, as bypass grafting isrequired in roughly 30% of all patients requiring coronary arteryrepair, and is firmly established as the best treatment option forpatients with multi-vessel coronary artery disease and diabetes. Thegold standard for sutures in vascular repair is polypropylenemonofilament (Prolene®) or expanded polytetrafluoroethylene (ePTFE,Gore-Tex®) sutures. These sutures are stiff and structurally dissimilarto native tissue, with no ability to deliver a sustained dose ofantiproliferatives. When a vein graft and artery are surgically joinedvia the clinically-favored “running” suture technique, there is asignificant reduction in elasticity at the suture line, primarily due tothe stiffness of industry sutures compared to the natural elasticity ofthe adjoining vessels. This stiffness is postulated to exacerbate IHthrough various mechanisms, suggesting there could be an improvedhealing response to a suture with better circumferential compliance(elasticity) at the suture line. Dissolvable sutures have also beendeveloped for microvascular anastomoses, but the inherent loss ofmechanical strength over time, increased cytotoxicity from degradationfactors, and the risk of dislodged suture particles forming an embolismare too great to justify their study in a clinical setting. Alternativesto sutures have also been explored, but further work is needed beforethey are applicable for routine use.

To address the significant need to reduce IH in vascular suture repairs,we propose a suture that will: a) locally deliver a naturally occurringSMC-specific antiproliferative agent, b) better match the elasticity ofthe adjoining vessels, and c) encourage natural long-term healing due toits nanofibrous morphology.

Composition to address problem: Polymer, Polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT)),polyurethane or combination of polyester and polyurethane; BioactiveAgents: Anticoagulant (recombinant hirudin or Argatroban),antiproliferative (paclitaxel, everolimus, sodium butyrate and/orsilencing siRNA), growth promoting (vascular endothelial growth factor,fibroblast growth factor) and/or antimicrobials (antibiotics,antimicrobial peptides, naturally-occurring antimicrobial proteins);Shape: Yarn-like construct (nanofibrous single strand yarn); Dimensions:Thickness can be varied (0.025 mm-2 mm); length can be varied from1m-2m. Can be made in a continuous fashion.

Synthesis Procedure: A polymer solution of 7% (w:v) polyurethane (cPU)and 3% (w:v) polyester (PTT; 0.63 dl/g) was prepared in HFIP and used tosynthesize cPU-PTT control sutures. Another cPU-PTT solution containing0.25% (w:v) Evero (LC Laboratories) was also prepared. Solutions weremixed for 48 hours prior to use. The collecting surface was acustom-designed apparatus consisting of a single 60 cm long, 0.75 mmdiameter Teflon-coated flexible stainless steel mandrel, bent into aring, grounded by a wire to the central axle, secured at both ends to amechanized base to create a torus configuration. This device ensures aneven coating onto the surface of the torus, while also creatingalignment of polymer nanofibers in the toroidal/lengthwise direction ofthe electrospun coating. This collecting surface was inserted into acustom-designed, computer-automated electrospinning unit. Sutures wereelectrospun for 5 minutes using a 3 ml/hour flow rate. +20 kV appliedvoltage and 15 cm gap distance. After electrospinning, the tubularnanofibrous material was removed, manually twisted and elongated to itsyield strain (300% of its original length) to create a suture. Eachsuture was then tightly coiled around a spool and placed into a vacuumoven (99.9% vacuum; 40° C., 24 hours). This process facilitatesvaporization of residual HFIP while also increasing tensile strength asa result of cold working, annealing, and radially contracting thefibers.

Wound Dressing/Pak

Uncontrolled bleeding (hemorrhage) continues to be the leading cause ofdeath upon deployment of military personnel in a theater of operations.Hemorrhage is also the second leading cause of death among civiliantrauma deaths. These numbers have remained very high all throughout thehistory despite numerous advances in emergency treatment for severelyinjured military personnel and for civilian trauma cases. Studiesindicate that more effective methods could have reduced mortality ratesfor our military personnel by at least one third. It is generallyaccepted that mortality-rates can be reduced considerably if thebleeding can be stopped within the first thirty-minutes of the trauma.As stated in the BAA “new materials and systems are required to advancethe medical capabilities currently available, thereby reducing theamount of preventable battlefield deaths and reducing the effectsresulting from injury.”

Hemostatic devices were one of the treatments developed to reducehemorrhage and save a soldier's lives. These devices are divided basedon the application type into four categories 1) powders/granular agents,2) solid materials, 3) flexible materials and 4) barrier agents orself-expanding gels. Powders are poured into the wounded areas andmostly work by absorbing fluids and low molecular weight products in theblood, thereby increasing the localized concentration of clottingfactors and enhancing clot formation. The void remaining in technologyfor preventing wound hemorrhage on the battlefield is the rationalebehind this BAA solicitation.

A light weight bioactive wound dressing/pack has been developed thatprovides the following characteristics: (1) Stop the bleeding quickly (2minutes or less) and more efficiently at any point on the body (i.e.extremities or non-compressible wounds in the abdomen region) (2) Beeasily applied by either a medic or by the wounded soldier themselves(3) Prevent wound infection resulting from a non-sterile environment vialocalized delivery of an antimicrobial agent (4) Provide directpain-relief by controlled release of an analgesic agent (5) Beready-to-use and requiring no special preparation/training (6) Bebreathable to help wound healing and (7) Be stable under variousclimatic conditions for extended periods (−10° C. to 40° C.).

Composition to address problem: Polymer: Polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT)),polyurethane or combination of polyester and polyurethane; BioactiveAgents: Coagulant (thrombin), antimicrobials (antibiotics, antimicrobialpeptides, naturally-occurring antimicrobial proteins) and/or analgesic;Shape: Flat narrow material (dressing), rounded tampon shaped, or twoflat electrospun materials joined together via ultrasonic welding orheat selling and containing super-absorbent polymer in the mid-portion;Dimensions: Variable width, length and thickness. In one embodiment, thewidth and length are each about 1 cm.

Synthesis Procedure: A nanofibrous bioactive hemostatic device prototypehas been developed using electrospinning technology. Unlike any otherhemostatic wound dressing present in the market, advanced wound dressing(AWD) has two components that work in a synergistic fashion to provide amulti-purpose hemostatic device. The first layer immediate to the woundcontains an active agent that rapidly promotes blood clotting. Thesecond layer has antibiotic and analgesics to ease pain, aid recoveryand prevent harmful life threatening infections. The materialscomprising these layers are made of polyester (PET). The PET polymer wasselected due to its inertness, ease of electrospinning with drugs, easeof surface modification, soft feel, toughness and flexibility of thefinal electrospun product. The resulting electrospun PET material isnon-toxic, porous (porosity=60%), nanofibrous (fiber diameter 300 nm to3 vm), anisotropic (have same properties throughout), permeable(improves breathability) nonwoven structure with a very high surfacearea to volume ratio (improves the attachment of active moieties andenhances the contact interaction of injured tissue). These qualities areespecially important for a wound dressing especially when dealing withnon-linear, deep, incompressible wounds where the dressing needs to bepacked into the wound. The blood-contacting layer is electrospun PETwhich is further mollified to create reactive groups along the surfaceof the nanofibrous layer. These functional groups are utilized to bind apotent coagulation enzyme onto the surface of the dressing. Thispro-coagulant is adsorbed electrostatically onto the surface ofsurface-modified electrospun PET, stabilizing the coagulant forlong-term storage. The coagulant is immediately released locally withinthe wound upon contact with blood providing rapid clot formation.Electrospinning also allows incorporation of selected drugs (antibioticand analgesic agents) which help in the healing process. These not onlyretain their properties but also are released at a sustained rate asshown in benchtop assays. While the selected blood coagulant proteinaccelerates the wound clot formation, the antibiotic and analgesicagents prevent bacterial infection while casing wound pain,respectively. The AWD can be cut into different geometries to treatwounds of various types and locations just like standard gauze.

The coagulant is a proven non-immunogenic natural enzyme that directlyactivates the coagulation pathway as compared to other indirectcoagulation drugs, chemicals or additives. The antimicrobial agent is abroad spectrum and third generation drug that is effective against awide range of gram positive and gram negative bacteria encountered inthe field under various combat scenarios. The analgesic agent is also apotent drug which is presently a part of the medic kit. This will be thefirst time to our knowledge that alt these agents will be delivereddirectly through a single hemostatic wound dressing.

Nanofibrous Stent Coating

Problem: Abnormal proliferation of neointimal smooth muscle cells (SMCs)is central to lesions of atherosclerosis and restenosis. Metallic stentdevices, with either a bare metal surface (BMS) or drug-eluting surface(DES), have become widely utilized as a first option for patients withdiseased blood vessels in which flow has been significantly restricteddue to this proliferative event, thereby compromising organ or limbfunction. Stents are preferred over standard surgical interventions suchas vessel bypass due to less invasiveness of the procedure andaccelerated patient recovery times. Unfortunately, restenosis ratesafter BMS placement range from less than 10% to as high as 58%, asignificant problem based on the 1.1 million stents annually implanted.Additionally, BMS have also been prone 10 late-term thrombosis andthromboembolism formation.

The advent of DES has reduced restenosis and stent thrombosis (ST) ratesas compared to bare metal stents (BMS). While it was predicted that DESwould overcome all of the complications associated with BMS, this hasnot come to fruition. The prevalence of these complications hasstimulated the development of the next generation of drug-eluting stentswhich are bioresorbable (BRS). However, these studies are relativelyshort-term, with long-term efficacy still undetermined. Regardless ofthe overall design (BMS, DES or BRS), stent use still focuses on twospecific criteria: 1) administration of systemic anti-platelet therapyand 2) delivery of a non-targeting anti-proliferative agent.Anti-platelet therapy is required in order to prevent thrombus formationon the stent until healing occurs. For anti-platelet therapy, asignificant shift in the length of time for systemic anti-platelettherapy from 1 month minimum delivery to a now recommended minimum 1year period has been implemented in order to drive down ST rates whileattempting to allow healing to occur. This treatment is being carriedout at a significant risk in hemorrhagic complications to the patient.Additionally, any deviation in anti-platelet therapy administrationsignificantly increases the risk of ST. Delivery of anti-proliferativeagents, while effective at preventing SMC proliferation, also affectsendothelial cells, resulting in delayed re-endothelialization andvascular inflammation. This lack of complete healing increases the riskof thrombus formation, resulting in the need for long-term systemicanti-platelet therapy. Bioresorption of the BRS stent may eliminate theneed for long-term anti-platelet therapy but issues with incompletehealing due to local delivery of these antiproliferative agents willstill persist. Thus, there is a need to investigate alternative stentcoating methodologies in which: 1) the effects of the treatments willpersist for an extended period of time, 2) drug delivery can be targetedto specific areas within the vessel, 3) a combinatorial approach interms of drug delivery can be used and 4) a scaffold for controlledvessel healing is provided.

Composition to address problem: Polymer: Polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT)),polyurethane or combination of polyester and polyurethane; BioactiveAgents: Anticoagulant (recombinant hirudin or Argatroban),antiproliferative (paclitaxel, everolimus, sodium butyrate and/orsilencing siRNA) and/or, growth promoting (vascular endothelial growthfactor, fibroblast growth factor); Shape: Uniform thin coating ofmetallic stent; Dimensions: Thickness can be varied (0.05 mm-0.30 mm) aswell as overall length.

Synthesis Procedure: A polyester (17.5% PET and 2% poly(butyleneterephthalate) or PBT; w:v) polymer solution was prepared in ice-cold100% hexafluoroisopropanol (HFIP) and mixed on an inversion mixer for 48hours. This PET solution was then diluted 50% with HFIP, mixed for 1hour and split in half. To one portion of this solution, 50 of DyLight550 (DyLight; 10 mg/ml) was added and mixed for 1 hour on an inversionmixer. During this time, a 2 mm internal diameter metallic stent(Medtronic, Inc.) was slid onto a 2 mm Teflon-coated stainless steelmandrel. Both polymer solutions (with and without DyLight) were loadedinto 5 ml syringes and placed onto our computer-automatedelectrospinning apparatus. The specific stent/mounting mandrel was setat a jet gap distance of 15 cm from the tip of the needle and theperfusion rate set at 3 ml per hour at 25° C. Perfusion of the polymerwith DyLight was started upon application of the current (+15 kV) withelectrospinning proceeding for 3 minutes. Perfusion of the unmodifiedPBT solution was begun immediately from another perfusion pump. Thissolution was electrospun for 5 minutes. Coated stents were thenair-dried in a vacuum oven (600 mm Hg 37° C.) for 48 hours to removeresidual HFIP.

Dermal Substitute/Artificial Skin Scaffold

Problem: Approximately 20% of cutaneous wounds with significant tissueloss transition into a non-healing or chronic state. Millions ofindividuals are impacted by pressure ulcers, including 600,000 patientswhose wounds are secondary to venous insufficiency, and up to 3 millionpatients whose wounds result from immobility. The incidence of thesetypes of chronic wounds, most prevalent in the elderly demographic, willlikely increase as the average age of the population rises. The totalcost of treating chronic wounds is estimated to exceed $8 billionannually, presenting a significant health care need. Beyond tissuedamage, infection is the most prevalent complication in chronic anddiabetic ulcers. Millions of clinical infection cases each yearworldwide, contributing to thousands of deaths, are attributed to twospecies of bacteria, Pseudomonas aeruginosa and Staphylococcus aureus,The growing number of injuries and pathologies has significantlyincreased demand for wound management products.

A critical factor in preventing infection, scarring, and death fromsevere skin wounds is the prompt restoration of skin integrity.Split-thickness autografts are considered the “gold standard” fortreating localized skin injuries, but the lack of available donor sitesand donor site morbidity routinely hinder the recovery of patients withchronic wounds. Further, as the wound healing process has already beencompromised in these patients with chronic wounds, autologous grafts maynot be appropriate. While several composite skin and dermal substituteshave achieved some clinical success for restoring damaged skin, limitedmechanical stability, sub-optimal wound healing, infection, scarring andprolonged healing times remain persistent problems. As such, there is acontinued need for a dermal scaffold that promotes rapid tissueregeneration and vascularization (cellular ingrowth and angiogenesis),maintains the mechanical stability of the tissue, and provides barrierfunction (microbial resistance) for the wound site.

Composition to address Problem: Polymer: Top Layer=Polyester(combination of polyethylene terephthalate (PET) and polybutyleneterephthalate (PBT)), polyurethane or combination of polyester and polyurethane. Bottom Layer comprises a biodegradable polymers(polycaprolactone, polyglycolic acid and/or polylactic glycolic acid);Bioactive Agents: Growth promoting (vascular endothelial growth factor,fibroblast growth factor) and antimicrobials ((antibiotics,antimicrobial peptides, naturally-occurring antimicrobial proteins);Shape: Flat sheet with non-degradable layer on one side and a degradablepolymer on the other Dimensions: Overall thickness can be varied (0.1mm-0.5 mm) as well as overall length.

Synthesis Procedure: Two polymer solutions were prepared in ice-cold100% HFIP. The first solution prepared contained a mixture ofpolyurethane (PU) and polyethylene terephthalate (PET) polymers (7% and3% w:v, respectively) with an antimicrobial agent. The second solutionwas composed of a mixture of polycaprolactone (PCL) and poly glycolicacid (PGA) polymers (15% and 5% w:v, respectively) with an antimicrobialagent and growth promoting factor. Both solutions were mixed for 48hours on an inversion mixer. A self-contained computer-animatedelectrospinning apparatus was utilized for electrospinning. A stainlesssteel 58-gauge blunt spinneret (0.5 mm internal diameter) was connectedto the polymer-filled syringe. The collecting surface (mandrel) was setat a jet gap distance of 15 cm from the tip of the needle. The perfusionrate was set at 3 ml per hour at 25° C. Perfusion of the polymer wasthen started upon application of the current to the tip of the needle(15-20 kV) with electrospinning proceeding. The PU-PET solution, whichwas loaded into a 10 ml syringe, was first electrospun onto a rotating35 mm cylindrical mandrel for 90 minutes (nPU-PET). Residual HFIP on theresulting nPU-PET material was removed via sonication of the material in100% ethanol for 30 minutes following by a sonication in water for 2minutes. After 48 hours of drying, the PCL-PGA solution was electrospunonto the pre-existing nPU-PET polymer sheet on the mandrel, yielding thecomposite dermal scaffold (nPCL-PGA layer). This composite material wasthen vacuum-dried (600 mm Hg) at 36° C. for 24 hours to remove residualHFIP from the nPCL-PGA layer.

Bone Fixation Coating Pins

Problem: External bone fixation is a method of aligning broken boneswhen more conventional methods (casting, internal fixation) areprecluded. In this method, pins or wires are inserted into the bone andanchored by a rigid external frame to maintain proper orientation of thefracture. This can be accomplished for temporary-stabilization pendingdefinitive care, or can be used over a prolonged period to stabilize thefracture until union occurs. In addition, external fixation can be usedfor many clinical applications including limb lengthening, deformitycorrection, and bone transport for treating critical sized bone defects.The most common complication of external fixation is pin site infection,with reported infection rates ranging dramatically in the literaturefrom 11-52%. Occurrence of these pin site infections is so common thatthey have become an accepted factor of external fixations in thehealthcare industry. The entry point of a bone pin is essentially achronic open wound, because the penetration of the pin interrupts theskin's ability to heal. These open wounds are highly susceptible tobacterial colonization and infection. External fixation pins can thusbecome colonized, providing a point of entry for bacteria to the softtissues and the bone itself. This exposure of open wounds to foreignbodies provides an ideal environment for bacterial adhesion andsubsequent biofilm formation, encouraging infection of adjacent tissues.

The current standard of treatment is for orthopedic surgeons toprescribe preventative pin care routines for the patient (e.g. dailybacitracin sponge washes, hydrogen peroxide washes), and to later dependon systemic and topical antibiotics when those measures fail. However,once bacteria adhere to the surface of the pin, they become highlyresistant to antibiotics, allowing some infections to persist despiteaggressive antibiotic treatments. The use of antibiotics that becomesnecessary in these cases contributes to the development of resistantbacteria including methicillin-resistant Staphylococcus aureus (MRSA),and could be mitigated by a localized prophylaxis at the pin site. Pinsite infections are costly, as they delay healing, prolong treatmenttime, and require repeat surgeries and aggressive antibiotic regimens.In many instances, the presence of persistent pin infections requiresremoval of the frame itself, potentially before the treatment of theunderlying fracture is complete. Yet, the need for a clinically proveninfection-resistant bone pin remains unanswered, and the infection ratefor external bone fixations remains high. With more than $15 billionspent on orthopedic infections each year, there is a significantclinical need to impart infection-resistance to bone pins.

Composition to address Problem: Polymer Polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate (PBT)),polyurethane or combination of polyester and polyurethane. BioactiveAgents: Antimicrobials ((antibiotics, antimicrobial peptides,naturally-occurring antimicrobial proteins); Shape: Nanofibrous coatingonto metallic bone pin; Dimensions: Overall thickness can be varied(0.05 mm-0.2 mm) as well as overall length.

Synthesis Procedure: A polymer solution comprising polyethyleneterephthalate (PET) with polybutylene terephthalate (PBT) (17.5% and 2%w:v, respectively) was prepared in ice-cold 100% HFIP and inversionmixed for 48 hours. This solution was then halved, and one half was keptunchanged to act as a control solution, while the other half was given1.5% w:v of Gentamicin and mixed for another 24 hours. A self-contained,computer automated electrospinning apparatus was utilized to coat thebone pin. This apparatus can electrospin onto cylindrical constructs,such as bone pins. A stainless steel 18-gauge blunt spinneret (0.5 mminternal diameter) was connected to the polymer-filled syringe. Onesmooth and one surface roughened (via sandblasting) 5 mm diameter, 316Lstainless steel rod (same material as a commercial bone pin) waspositioned in a chuck to rotate at 270 RPMs while the spinneret was setto traverse a 4 inches length of the rod at a rate of 2 inches persecond. This 4 inch length reflects the unthreaded portion of the actualbone pin (either left smooth, or sandblasted to improve adherence of thematerial) that would be coated. The rod was set at a jet gap distance of15 cm from the tip of the needle. The perfusion rate was set at 3ml/hour and +20 kV of voltage was applied to the spinneret at 25° C.Electrospinning of the polymer solution was then conducted for 10minutes on one 4 inch section of each mandrel, and for 20 minutes onanother 4 inch section of each mandrel. After the PET/PBT andPET/PBT/Gentamicin solutions were electrospun separately onto theirrespective 4 inch segments of cash rod, a 70° C. heat treatment at99.99% vacuum was applied for 24 hours to seal loose fibers down to thebulk material and to remove all of the residual solvent.

Pediatric Nanofibrous Catheter Cuff

Problem: In pediatric medicine, long-term treatment of oncological,hematological, and immunological disease requires the implantation of anindwelling central venous catheter (CVC). CVCs are necessary to deliverdrugs, nutritive fluids, chemotherapy, hemodialysis therapy, or to takeblood samples for testing without causing trauma to the patient. Yet,CVCs are prone to failure due to three primary mechanisms: infection(3-14%), dislocation (10-19%), and thrombosis (1-2%). Infections are theprimary concern with CVCs, since approximately 9-14% of pediatricpatients with CVCs contract catheter-related bloodstream infections(CRBIs), the mortality rate for which is over 13%. CRBIs in pediatricpatients have been reported to increase hospital stays by one week, andcost $39,219-$50,362 on average per infection, with an estimated 250.000CRBIs occurring in the US each year 8. The other major concern isdislocation, where the CVC is accidently pulled out from the originalsite of implantation. This occurs most frequently in pediatric patientpopulations because a child's tendency to pull on the protrudingcatheter tubing.

Bacterial infections of CVCs originate on either the catheter's externalsurface or within the luminal surface. Most infections and CBRIs arecaused by the migration of bacteria from the skin down the externalsurface of the catcher, although these infections can also migrate downto the internal lumen from the hub. Catheter infections in the lumen canbe eradicated using a simple procedure called “antibiotic-, orethanol-locks,” in which either a heparinized antibiotic solution or a70% ethanol solution is injected into the infected catheter lumen andheld there for several hours. Yet, there is no comparable way toeradicate an established bacterial infection on the external surface ofa catheter. Infection of the external surface requires complete removalof the CVC, possibly preventing the patient from receiving vital therapyor nutritive fluids. Thus, preventing the migration of bacteria from theskin is essential to reducing catheter infections and CRBIs.

Composition to address Problem: Polymer: Polyester (combination ofpolyethylene terephthalate (PET) and polybutylene terephthalate; (PBT)),polyurethane or combination of polyester and polyurethane; BioactiveAgents: Antimicrobials (antibiotics, antimicrobial peptides,naturally-occurring antimicrobial proteins); Shape: Nanofibrous coatingonto a catheter device; Dimensions: Thickness can be varied (0.025 mm-2mm); Coating length can be also varied.

Synthesis Procedure: Two polymer solutions, one containing 7% (w/v) ofpolyurethane 55D (PU) and 3% (w/v) polyethylene terephthalate (PET), andlater another containing 7% cPU (w/v) and 3% PTT (w/v) were prepared inice-cold hexafluoroisopropanol (HFIP). Both polymer solutions were mixedfor 48 hours on an inversion mixer. Each solution was halved, with onehalf of each solution kept unchanged to act as a control solution, and1.5% (w/v) of Ciprofloxacin (Cipro) was added to the other half of eachsolution before mixing all solutions for another 24 hours. Each solutionwas then placed into a 10 ml syringe for electrospinning. Aself-contained, computer-automated apparatus was used to electrospin thematerials. To create the materials tested in PS 1-3, a segment ofpolyethylene catheter tubing (14 cm length) with an inner diameter of1.57 mm and outer diameter at 2.08 mm was used as a base material ontowhich the PU-PET solutions were electrospun. To create the materials,cPU-PTT solutions were electrospun directly onto a 35 mm diametermandrel. Electrospinning parameters used for all nanofibrous cathetercuffs were: gap distance of 15 cm, applied voltage of 15 kV, perfusionrate of 3 ml/hour and electrospinning times of 5, 15, and 30 minutes(n=2 segments/electrospin time/solution). After electrospinningnanofibrous (n-)PU-PET control and Cipro-loaded cuffs, residual HFIP wasremoved by a sonication in ethanol for 30 minutes followed by asonication in deionized water for 2 minutes. HFIP was removed fromncPU-PTT control and Cipro-loaded cuffs using a vacuum oven at 37° C.(99% vacuum) for 24 hours.

Other Commercial Applications

In other embodiments, nanofibrous materials with increased water moving(wicking) properties are provided. Polyester polymers are electrospun asdescribed elsewhere in this specification. The materials are chemicallytreated (sodium hydroxide or ethylenediamine treated), resulting insurface functional groups. The materials can be used in variousconstructs for different applications that require water/solutionmovement.

In other embodiments, nanofibrous materials are provided to treatfinger/toe nail and yeast infections (anti-fungal delivery). Polyesterpolymers containing antifungal agents are electrospun as describedelsewhere in this specification. The resulting materials are cut andadhered to artificial finger nail or can be formed into its own device(nail coating/tampon device).

In other embodiments, nanofibrous materials with radiopaque propertiesare provided. Polyester, polyurethane and/or a polyurethane/polyestercombination with radiopaque agents (diatrizoic acid, barium sulfate) maybe synthesized as described elsewhere in this specification. Thematerials are useful for sutures, device location and wound dressing.

In other embodiments, nanofibrous materials are used as filtrationdevices. Polyester polymers are electrospun as described elsewhere inthis specification. The materials are chemically treated (sodiumhydroxide or ethylenediamine treated), resulting in surface functionalgroups. Specific bioactive moieties can be immobilized to this materialand used as a filtration medium to remove targeted agents.

Series A: Preparation and Characterization of Nanofibrous (nPET)Textiles Experiment 1. The Electrospinning Perfusion Technique TheElectrospinning Apparatus

For small batch purposes, a computer-automated electrospinning perfusionapparatus was assembled which included a power supply, a syringe pump,an elevated holding rack, a modified polyethylene chamber, a spray headwith power attachment, a reciprocating system, and a stirrer forcontrolled mandrel rotation. Such an assembly is shown by FIG. 4.

Utilization of this assembly permits uniform coating of a liquid polymeronto the PTFE-coated stainless steel mandrel (diameter=0.75-40 mm). A 10ml chemical-resistant syringe was filled with the liquid polymer; and astainless steel 18 gauge blunt spinneret (0.5 mm internal diameter) wascut in half, with the syringe fitting half connected to thechemical-resistant syringe.

Nalgene PVC tubing ( 1/32 ID.times. 3/32 OD; 66 cm length) was thenconnected to the syringe, followed by connection to the other half ofthe blunt spinneret within the spray head. The line was purged of air,with the syringe then placed onto the syringe pump. The high potentialsource was connected to the spray head tip; and the mandrel was set at ajet gap distance of 15 cm from the tip of the needle. The mandrel wasthen grounded to the power source; and the perfusion rate was set at 3ml/hour at 25° C.

The Polymer

A polyethylene terephthalate (20% w:v) polymer was prepared in ice-cold100% hexafluoroisopropanol. The 10 ml syringe with a stainless steel18-gauge blunt spinneret (0.5 mm internal diameter) was filled with thesolution and placed onto the Harvard Apparatus syringe pump.

The Perfusion Technique

Perfusion of the polymer was then started upon application of thecurrent to the tip of the needle (15 kV) with electrospinning proceedingfor 40 minutes. After electrospinning, the end portions of the resultingtubular structures comprised of nanofibrous polyethylene terephthalate,now termed “nPET” structures, were cut off and discarded (1 cm from eachend of the mandrel). The original nPET tubular structures were thenstretched 25% of the starting segment size while on the mandrel in orderto provide a set stain across the fibers, a process that occurs innormal fiber extrusion. This yielded sized tubular segments of nPETfabric.

Some, but not all, of the stretched nPET segments were then immediatelyexposed to 100% ethanol for 2 hours at room temperature (or for 30minutes in 100% ethanol with sonication) in order to remove the residualsolvent. Then, all of the nPET tubular structures (ethanol exposed ornot) were air-dried overnight at room temperature.

Results

The nPET tubular segments, whether air-dried or exposed to ethanolfollowed by air-drying, had a consistent 4 mm internal diameterthroughout the lumen (length=7.5 cm). A total of 4 nPET structures weresynthesized for each method using the above-described process.

For this experimental study, the nPET segments air-dried at 60° C. wereemployed for all of the subsequently conducted in-vitro studies reportedherein. This post-synthesis treatment was performed owing to thepossibility of Cipro eluting during the ethanol incubation for the othermethodology described later herein.

Concerning the electrospinning technique itself for tubular structuresfabricated using the described parameters, it was found that increasingelectrospinning time significantly beyond 40 minutes increased therigidity of the resulting nPET material. Conversely, electrospinning theliquid polymer blending for shorter periods of time (e.g., 1-15 minutes)provided a tubular structure without significant (less than 1 poundbreak strength) wall strength. Major differences in and variance oftubular wall rigidity may be desired for the various medical articlesand devices to be employed clinically. However, the chosen parametersemployed for nPET material formation in these experimental studies wereuniformly and consistently maintained at 40 minutes of electrospinningtime, a polymer concentration of 20%, an applied voltage (15 kV), and agap distance of 15 cm.

Experiment 2: Characterization of Physical Properties of ElectrospunnPET Material Tensile Strength/Ultimate Elongation

Tensile strength (pounds force), strain at maximum load (%) and strainat break (%) for knitted DACRON segments (formed of a commerciallyobtained standard textile material) and for electrospun nPBT segments(formed of a polyethylene terephthalate compound prepared as describedabove) were measured using previously published techniques. Control andtest segments (7 mm width, 3 cm length; n=3/test condition) of bothkinds of material were measured and cut.

A Q-Test Tensile Strength Apparatus (MTS Systems, Cary, N.C.) wascalibrated according to manufacturer's specifications in aclimate-controlled environment (room temperature=70° F., 65% relativehumidity). Each of the samples under test were also conditioned in thisenvironment for 24 hours. Segment stretching (crosshead speed=50 mm/min.gauge length=2 cm, load cell=25 lb) was then initiated and terminatedupon segment breakage.

Results

There was a marked difference between the break load of knitted DACRONsegments (42±9 pounds force) and electrospun nPET segments (3.7±0.9pounds force). This difference in breaking load was expected owing tothe significantly greater wall thickness of the knitted DACRON material.The other physical properties, such as the percent strain al maximumload (60±24 versus 55±8) and percent strain at break (60 versus 62±3),were comparable between the two test materials, indicating that thedifference in break strength was directly related to wall thickness.Thus, the nPET material is shown to possess significant physicalcharacteristics that would permit its presence and application invarious medical devices.

Experiment 3: Evaluation of Electrospun nPET Material Via ScanningElectron Microscopy Scanning Electron Microscopy (SEM)

Two electrospun nPET segments were randomly selected and examined via aJBOL JSM 5900 LV electron microscope in order to determine fiber sizeand distribution throughout the material wall.

Results

Analysis of electrospun nPET tubular structures via SEM revealed thatthe diameter of the polyethylene terephthalate fibers comprising thenanofibrous material varied from about 100 nm to 3000 nm in size. Thisis shown by the microphotographs of FIG. 5A and FIG. 5B. A comparisonSEM analysis of the knitted DACRON samples revealed that the knittedDACRON fibers ranged from 15 to 30 micrometers in diameter size (datanot shown) and thus were significantly larger than the nPET fiberdiameter size range.

Series B: The Agent-Releasing Textiles Comprising the Present InventionExperiment 4: Synthesis of Novel nPET Materials with Biologically ActiveAgents

Prior to forming the blended polymer solution, the solubility of Cipro,Diflucan and Paclitaxel in the HFIP (hexafluoroisopropanol) solvent wasdetermined. Based on the pre-chosen concentration of active agent to beemployed in the composite, 15 mg of each respective agent was placedinto 1 ml of the HFIP solvent, mixed and observed.

Following this initial assessment, polyethylene terephthalate (19%)polymer solutions containing either Cipro, or Diflucan, or Paclitaxel(1.5% w:v) respectively were prepared in ice-cold 100%hexafluoroisopropanol. These individually prepared polymer solutions ofCipro, or Diflucan, or Paclitaxel were mixed on an inversion mixer for48 hours in order to completely solubilize both the polyethyleneterephthalate polymer and each active agent component in theirrespective individual solutions. Then, the self-contained,semi-automated electrospinning apparatus (described previously herein)was again employed for fabricating each version of nanofibrous textilematerial.

Utilization of this system permits uniform coating of the preparedpolyethylene terephthalate polymer solution onto the PTFE-coatedstainless steel mandrel (diameter=4 mm). Using the uniform set ofparameters of the previously described experimental series, the mandrelwas set at a jet gap distance of 15 cm from the tip of the needle. Themandrel was then grounded to the power source. The perfusion rate wasset at 3 ml/hour at 25° C. Perfusion of the polyethyleneterephthalate/active agent mixture was then started upon application ofthe current to the tip of the needle (15 kV) with electrospinningproceeding for 40 minutes. After electrospinning, the end portions ofthe original tubular structure (1 cm from each end of the mandrel) werecut off and discarded. This resulted in textile tubular segments offixed length.

The resulting tubular segments were then stretched 25% of the startingsegment size while on the mandrel in order to provide a set strainacross the fibers, a process that occurs in normal fiber extrusion.These tubular segments were then either air-dried at 60° C. overnight;or exposed to 100% ethanol for 2 hours at room temperature in order toremove the residual solvent. Due the fluorescent properties of Cipro,nPET segments (those having no active agent) and nPET-Cipro segments(those having Cipro as the active agent)—having been already exposed to60° C. temperature overnight or to 100% ethanol for 2 hours—were thenexposed to a hand-held UV light to qualitatively assess Cipro presencewithin the textile structure.

Results

Cipro, Diflucan and Paclitaxel individually were each found to haveexcellent solubility in the HFIP solvent. Once combined with thepolyethylene terephthalate polymer/HFIP liquid, the solubility of eachrespective active agent remained unchanged. Formation of nPET (as asubstantive material) and of nPET tubular structures containing eitherCipro, or Diflucan, or Paclitaxel were all successfully accomplished.All these structures showed a consistent 4 mm internal diameterthroughout the lumen for each tubular structure (material length=7.5cm). Based on the perfusion rate in conjunction with electrospinningtime, each tubular segment incorporated approximately 30 mg of eachrespective active agent.

In addition, similarly to our previous experimental series, increasingelectrospinning time significantly increased the rigidity of theresulting nanofibrous material. Conversely, electrospinning for shorterperiods of time (1-15 minutes) provided a tubular structure withoutsignificant wall strength.

Furthermore, gross observation of the various resulting tubular segmentsvia UV illumination revealed intense fluorescence from the nPET-Ciprosegments, whether air-dried or ethanol washed, when compared to the nPETsegments. This UV illumination data demonstrated the presence of Ciproto be only within the nPET-Cipro segments. This effect is illustrated byFIG. 6.

Experiment 5: Determination of Cipro and Diflucan Release fromnPET-Cipro and nPET-Diflucan Segments Via UV/VIS SpectrophotometerMethods

nPET segments, nPET-Cipro segments, and nPET-Diflucan segments (0.5 cmsegment length, n=3 segments/time interval/segment treatment) wereindividually placed into 5 ml of phosphate buffered saline (PBS)followed by continuous agitation using Rugged Rotator inversion mixer(33 r.p.m.) at 37° C. Wash solutions were sampled at acute (0, 1, 4 and24 hours) and chronic (2-21 days for Cipro and 2-7 days for Diflucan)time periods, with replacement of the wash solution with a fresh 5 mlPBS after sampling. The absorbance of wash solutions were read at 322 nm(PBS blank) using a Beckman DU640 UV/VIS spectrophotometer.

A standard curve using known Cipro concentrations ranging from 0-100micrograms per ml was prepared. This Cipro standard curve was then usedto extrapolate the antibiotic concentration within the wash solutions.

Results

The release profiles for the nPET-Cipro segments are shown by FIG. 7,and the release profiles for the nPET-Diflucan segments are shown byFIG. 8. Notably, the release profiles for each type of segment aremarkedly different.

As observed and recorded, Cipro release within the first 4 hours wasconsistent at 5±2 micrograms per ml, and was followed by a sharpincrease in rate to 13±4 micrograms per ml at 24 hours. Cipro releasethen decreased to 6±4 micrograms per ml by 48 hours, but persisted(ranging from 1-2 micrograms per ml) throughout the time duration ofthis study (504 hours). The amount of Cipro released has significantbiological activity, owing to the low MIC₅₀ for Cipro (0.26 microgramsper ml).

In comparison, Diflucan release followed typical first order kinetics inthat the greatest release occurred within the first 24 hours (17, 12 and11 micrograms per ml, respectively). This was followed by a slowsustained release over the remaining time periods over the 168 hourstudy period, the time duration of this study.

Overall therefore, nPET segments containing Cipro and Diflucandemonstrated significant release of each active agent throughout thetime periods empirically evaluated.

Experiment 6: Antimicrobial Activity of nPET Segments and nPET-CiproSegments Via a Zone of Inhibition Assay Methods

nPET segments (n=3 segments/time interval) and nPET-Cipro segments (n=9segments/time interval), which were previously washed as describedabove, were then evaluated for antimicrobial activity using a zone ofinhibition assay.

A stock solution of S. aureus was thawed at 37° C. for 1 hour. Uponthawing, 1 microliter of this stock was added to 5 ml of Trypticase SoyBroth (TSB) and incubated overnight at 37° C. From this solution, 10microliters was streaked onto Trypticase Soy Agar (TSA) plates. nPETsegments and nPET-Cipro segments were individually embedded into the S.aureus streaked TSA plates; and each prepared plate was then placed intoa 37° C. incubator overnight. Standard 5 micrograms Cipro Sensi-Discs(n=3) were also embedded into the S. aureus streaked TSA plates at eachtime interval as a positive control. The zone of inhibition each piecewas determined, taking the average of 3 individual diametermeasurements. Zone size (mm) over time was determined for eachparameter. The prepared assay plates are illustrated by FIG. 9.

Results

The nPET-Cipro segments demonstrated significantly greater antimicrobialactivity than nPET segment controls at all of time periods examined.This is graphically shown by the data of FIG. 10.

The zone of inhibition created by the 5 micrograms Cipro Sensi-Discs wasconsistent at 23 mm. The nPET-Cipro segment antimicrobial activityprofile correlated with the Cipro release determined in thespectrophotometric studies—in that the greatest antimicrobial activityoccurred within the first 48 hours. Cipro antimicrobial activity,presumably caused by lower Cipro concentrations being released over timeas determined by the spectrophotometry, decreased slowly over theremaining time periods. Nevertheless, significant antimicrobial activitywas still evident even after 504 tours, with inhibition zones beingcomparable to those of the Sensi-Disc results. Thus, this studydemonstrates that Cipro release from the nPET material persisted forover 504 hours, with antimicrobial activity correlating to the quantityof Cipro release.

Experiment 7: Anti-Fungal Activity of nPET Segments and nPET-DiflucanSegments Using a Turbidity Assay Methods

Candida albicans was purchased from ATCC. The fungus was re-hydrated inYM Broth with 0.5% dextrose and grown for 30 hours at 30° C. underhumidified conditions. nPBT segments and nPET-Diflucan segments (1square cm, n=2 segments/inoculum/treatment) were prepared as previouslydescribed herein, and then tested against various Candida albicansconcentrations.

A broth macrodilution assay was performed based on the NCCLS M27-Aprotocol. The stock fungal inoculum concentration was determined viabackplating a set volume of the diluted fungus broth onto Trypticase SoyAgar plates. The number of colony forming units (cfu) grown per platewas then counted and extrapolated to determine the starting Candidaconcentration.

The stock fungus solution was then diluted to 10⁶, 10⁵ and 10⁴ cfu/ml.After incubating the individual test segments in 2 ml of the fungussolutions for 24 hours at 30° C., the optical density of the brothsolutions was measured at 492 nm. These values were compared to Candidasolutions without any nPET materials (serving as the positive control)as well as against YM Broth only and Candida solutions with 40micrograms Diflucan solution (both serving as negative controls).

Results

The nPBT-Diflucan segments had significantly greater antifungal activityat all wash periods as compared to nPET segments which had no antifungalactivity (turbidity comparable to Candida control). This is graphicallyshown by the data of FIG. 11.

Diflucan (40 micrograms) in solution demonstrated excellent antifungalactivity against this inoculum, with decreasing activity as the inoculumincreased. Antifungal activity by the nPET-Diflucan segments was clearlyevident at all Candida concentrations evaluated with activity mimickingsolution-based Diflucan (data not shown). Thus, this experimental studydemonstrated that Diflucan is released from the electrospun nanofibrousmaterial even after extensive washing for 2 days, with Diflucanmaintaining it recognized and characteristic antifungal activity aftersynthesis of the nPET-Diflucan tubular structure.

Experiment 8: Development of Electrospinning Methodology for Flat SheetNanofibrous (nPET) Material Methods

As described in Series A above, prepared polyethylene terephthalatechips were dissolved in ice-cold 100% hexafluoroisopropanol (19% w:v)and mixed on an inversion mixer for 48 hours in order completelysolubilize the chips. The self-contained, semi-automated electrospinningapparatus containing a Glassman power supply, a Harvard Apparatussyringe pump, an elevated holding rack, a modified polyethylene chamber,a spray head with power attachment and a reciprocating system was againused.

The stirrer was used to provide a holding chamber for the new flatcollecting plate employed to generate a sheet format. The design of thissurface is based upon the collecting plate. In short, a flat 12cm.times.10 cm copper plate, containing a 6 cm stainless steel rodextending from the underside of the plate was designed and grounded tothe power source.

A 10 ml chemical-resistant syringe was filled with the polymer liquid. Astainless steel 18-gauge blunt spinneret (0.5 mm internal diameter) wasthen cut in half, with the syringe fitting end connected to thepolymer-filled syringe. Nalgene PVC tubing was connected to the syringefilled with the polymer solution followed by connection to the otherhalf of the blunt spinneret within the spray head. The line was thenpurged of air, with the syringe then placed onto the syringe pump. Thehigh potential source was connected to the spray head tip, with theplate set at a jet gap distance of 15 cm from the tip of the needle. Theperfusion rate was set at 3 ml/hour at 25° C.

Perfusion of the polymer liquid was started upon application of thecurrent to the tip of the needle (15 kV) with electrospinning proceedingfor 1 hour and 40 minutes, with rotation of the plate 20° every 20minutes. This resulted in a flat, planar sheet of nPBT nanofibrousmaterial being formed. The resulting nPET sheet is illustrated by FIG.12.

After the electrospinning procedure was completed, a 1.0 cm marginaround the perimeter edge of the entire nPET planar sheet was cut off inorder to eliminate potential variability in the fabric thickness alongthe edge. The flat nPET sheet construct was then stretched 25% in thewidth and length of the material in order to provide a uniform setstrain across the fibers, followed by air-drying at 60 overnight.

Results

A flat sheet of electrospun nPET textile fabric (8 cm.times.10 cm) wasformed using this alternative method and technology. When viewed ingross, the nPET planar sheet had excellent handling characteristics andpossessed physical properties comparable to the nPET tubular structures.

VII. Conclusions Drawn from and Supported by the Empirical Data

1. The self-contained, semi-automated electrospinning apparatus providedby the present invention can be employed to generate two differentformats of nanofibrous textile fabrics. One format is a tubularstructure having determinable inner wall and outer wall diameter sizes,two open ends, and an internal lumen typically less than about 6millimeters in diameter. This tubular structure format presents aninterior wall surface and an exterior wall surface, and is a conduitbiocompatible with and suitable for the conveyance of liquids and gasesthrough its internal lumen.

A second format is a Hat or planar sheet construction havingdeterminable, length, width, and depth dimensions. The flat sheet fabriccan be folded and refolded repeatedly; can be cut and sized to meetspecific configurations; is resilient and can be prepared in advance toprovide varying degrees of flexibility, springiness, suppleness, andelasticity.

2. A wide range and variety of agent-releasing textiles can be preparedfor use as medical articles and devices using the present invention. Theagents are biologically active and well characterized; are incorporatedin chosen concentrations as an ingredient in the bulk polymer prior tomaking the textile fabric; and become indefinitely attached to andnon-permanently immobilized upon the fabricated nanofibrous textilematerial as a concomitant part of the process for manufacturing thetextile.

3. After being placed in a water containing environment, theagent-releasing textile will begin to take up water, release itsincorporated biologically active agent in-situ over time; and deliverthe release active agent at measurable concentrations directly into theadjacent and surrounding milieu. The in-situ released agent is function,operative and potent; and provides/performs its well recognized andcharacteristic biologically activity whenever and wherever it isdelivered.

While the invention has been described with reference to preferredembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof to adapt to particular situations without departingfrom the scope of the invention. Therefore, it is intended that theinvention not be limited to the particular embodiments disclosed as thebest mode contemplated for carrying out this invention, but that theinvention will include all embodiments falling within the scope andspirit of the appended claims.

What is claimed is:
 1. A method of forming a fabricated textilecomprising nanofibers by electrospinning perfusion, the methodcomprising the steps of: dissolving 19-20% weight per volume of anon-biodegradable polymer in hexafluoroisopropanol to provide anadmixture, the dissolving occurring at an ice-cold temperature, whereinthe non-biodegradable polymer is not polytetrafluoroethylene,polypropylene, or polyethylene; loading the admixture into anelectrospinning perfusion instrument which can be set at a specifiedflow rate; applying an electric current of 15-20 kV to a needle of theelectrospinning perfusion instrument; perfusing the admixture onto atarget surface at the specified flow rate, the step of perfusingoccurring at a temperature between about 20° C. and about 50° C. toprovide a perfused nanofiber having a diameter from 100 nm to 3000 nm;and permitting trace hexafluoroisopropanol to be removed from theperfused nanofiber to form a fabricated textile.
 2. The method of claim1, further comprising dissolving at least one biologically-active agentsuch that the admixture comprises a mixture of the non-biodegradablepolymer and the at least one biologically active agent.
 3. The method ofclaim 1, wherein the step of permitting trace hexafluoroisopropanol tobe removed is performed using a post-treatment process performed afterthe step of perfusing the admixture.
 4. The method of claim 1, whereinthe target surface is a mandrel.
 5. The method of claim 1, wherein thetarget surface is a metallic stent that is slid onto a mandrel andcoated with perfused material, and wherein the coated metallic stent isair-dried in a vacuum oven at 37° C. for 48 hours to remove residualhexafluoroisopropanol.
 6. The method of claim 1, wherein thenon-biodegradable polymer is selected from the group consisting of anon-biodegradable polyester, a polyurethane, and combinations thereof.7. The method of claim 1, wherein the target surface comprises a firstportion and a second portion and the step of perfusing the admixtureonto the target surface perfuses the admixture for a first period oftime onto the first portion and for a second period of time onto thesecond portion, wherein the first period and the second period aredifferent.
 8. The method of claim 1, the method further comprisingremoving the perfused fabricated textile from the target surface.
 9. Themethod of claim 4, wherein the perfused fabricated textile is formedinto a tubular construct.
 10. The method of claim 9, wherein the tubularconstruct has an internal diameter of at least 1 mm and less than 40 mm.11. The method of claim 9, wherein the tubular construct has a length ofat least about 1 cm and less than about 80 cm.
 12. The method of claim4, wherein the perfused fabricated textile coated mandrel is processedto remove traces of residual solvent and one edge of the perfusedmaterial is rolled towards an opposite end of the mandrel to achieve adesired thickness, and wherein the perfused material is cut along anopposite edge and the opposite edge is fused to form a rounded cuffshape.
 13. The method of claim 8, wherein the perfused fabricatedtextile is a flat sheet with a width of at least 1 cm and a length of atleast 1 cm.
 14. The method of claim 13, further comprising fusing theperfused fabricated textile to a second flat sheet comprising abiodegradable polymer.
 15. The method of claim 1, wherein the step ofdissolving further comprises dissolving a biodegradable polymer suchthat the admixture comprises a mixture of the non-biodegradable polymerand the biodegradable polymer.
 16. The method of claim 2, wherein the atleast one biologically-active agent is maintained at a temperature belowabout 50° C. during the steps of dissolving, loading, perfusing andpermitting such that the at least one biologically active agentmaintains the same biological activity after the method as the at leastone biologically active agent had before the method.
 17. The method ofclaim 1, wherein the target surface is configured to create alignment ofpolymer nanofibers in a toroidal direction with respect to a revolutionof the target surface.
 18. The method of claim 17, wherein the polymernanofibers are removed from the target surface and manually twisted andelongated to their yield strain to produce a single-strand yarn shape.19. The method of claim 18, wherein the single-strand yarn shape has adiameter between about 0.025 mm and about 2 mm.
 20. The method of claim1, wherein a 15-30 centimeter jet gap exists between the needle and thetarget surface.
 21. The method of claim 1, wherein a 15 centimeter jetgap exists between the needle and the target surface.
 22. The method ofclaim 1, wherein the fabricated textile is formed into a medical devicewithout the use of an underlying scaffold.